A novel Green systematic HPLC and UV-Spectroscopic method for the determination of rifaximin in tablet formulation

A novel Green systematic HPLC and UV-Spectroscopic method for the determination of rifaximin in tablet formulation | 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 Article A novel Green systematic HPLC and UV-Spectroscopic method for the determination of rifaximin in tablet formulation Saroj Kanta Bisoyi, Debasish Pradhan, sudhir Sahoo, Umashankar Mishra, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4394184/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 sustainable approach has been devised for quantifying rifaximin in pharmaceutical products, utilizing both spectrophotometric and HPLC methods. In the spectrophotometric technique, accurate measurement of absorbance at 296 nm detection wavelength was conducted by taking water and acetonitrile (1:1) as the solvent. For the HPLC method, a phenomenonex C18 250 x 4.6mm,5µ column was employed, with water and acetonitrile (1:1 v/v) serving as the mobile phase. Isocratic separation elution technique was employed at a rate of 1 mL per minute, with detection of rifaximin at 296 nm. These methods were found to be cost-effective, quick, eco-friendly, and straight forward, giving an analysis of rifaximin in 20 minutes. Application of these methods to pharmaceutical products yielded results free from matrix interference, with statistical comparison showing no significant differences among the techniques. Furthermore, the assessment of greenness conducted using AGREE software highlighted the developed methods' is environmental friendliness, which rely on solvents such as acetonitrile and water. These findings advocate for the adoption of our both analyst- and environmentally-friendly methods for the determination of rifaximin in tablet formulation over presently employed techniques. Rifaximin tablet eco-friendly RP-HPLC environmentally conscious UV-spectrophotometer Software AGREE assessment Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In the last few years, there has been a notable surge in interest among researchers towards the advancement of green analytical methods 1 – 4 . This heightened attention stems from a collective effort to mitigate effects on the environment and safeguard the well-being of analysts 5 – 8 . Reverse phase liquid chromatography (RP-HPLC) stands as a pivotal technique in drug analysis within the pharmaceutical industry, playing a crucial role in both product development and the quality control test of pharmaceutical formulations 9 – 13 . Typically, HPLC techniques rely on a stationary phase that is hydrophobic in conjunction with the help of polar mobile phase to achieve effective separation 14 – 16 . Commonly utilized column types such as C18 and C8 facilitate the separation of various components within the sample, thereby expediting HPLC studies, streamlining the analysis process, and ultimately reducing costs 17 – 20 . Optimal column selection is imperative for ensuring satisfactory chromatographic peaks and achieving robust separation of analytes 21 – 25 . In HPLC procedures, mobile phases are generally composed of a mixture of organic solvents such as acetonitrile and water, which is frequently enhanced with additives or buffer solutions to regulate pH. Because of its perfect miscibility with water, low viscosity, and limited chemical reactivity with device components and column surfaces, acetonitrile is a preferred option 26 – 30 . While factors like analysis time, accuracy, precision, and robustness have typically been given top priority in the development and validation of chromatographic methods, factors like the method's influence on the environment and the safety of analysts have historically gained less attention. This underscores the need for a more comprehensive consideration of these factors in method development and validation processes 31 – 33 . Modern antibiotics like rifaximin are essential for treating traveler's diarrhoea in those 12 years of age and older, especially when non-invasive Escherichia coli strains are the cause. Derived from the antibiotic rifamycin, which is well-known for having strong antibacterial activity despite having a low rate of gastrointestinal absorption rifaximin works by attaching itself to the beta-subunit of RNA polymerase that is dependent on bacterial DNA. The catalytic step that is necessary to polymerize deoxyribonucleotides into a DNA strand is effectively hampered by this mechanism. Rifaximin Physiochemical Properties given in table no-1 34–35 . Methodology Instrument A double beam spectrophotometer, the Shimadzu UV 1700, was used for spectrophotometric investigations that was manufactured by Shimadzu, Japan, and was managed by UV-Probe software. The experiments were conducted within the UV region scanning with 200.0–400.0 nm, with quartz cuvettes employed for sample containment. For HPLC analysis, an Agilent LC 1260 series system sourced from Agilent Technologies in the USA was utilized. Compound separation was accomplished with a Phenomenex C18 column (5.0 µm; 4.6 × 250.0 mm in dimensions) purchased from Agilent in the United States. Materials: All of the solvents used in liquid chromatography were gradiently pure. Rifaximin Active pharmaceutical ingredient as gift from pharmaceutical industries and Rifaximin tablets (550 mg) were acquired from a nearby drugstore for incorporation into this study. To prepare solutions along with the mobile phase, ultrapure water and acetonitrile was employed. Prior to analysis, the mobile phase underwent filtration using a 0.45 µm membrane filter under vacuum conditions and was subsequently sonicated. Preparation of standard solutions: For UV Spectroscopy Weigh accurately and transfer 10.34 mg of Rifaximin API into a volumetric flask with a capacity of 100 mL. To dissolve, sonicate the mixture after adding 50 millilitres of diluent. Add diluent to adjust the volume and stir. Further transfer 1.00, 2.00, 4.00, 6.00, 8.00 and 10.00 mL of standard stock transfer into 20 mL volumetric flask with volume added with diluents to attain the concentration of 5.16, 10.32, 20.63, 30.95, 41.26, 51.58 03 µg mL. For HPLC Accurately weigh and transfer 50.12 mg of Rifaximin API into a 100 mL volumetric flask. Add 50 mL of diluent to the mixture and sonicate to dissolve. To adjust the volume, add diluent and stir. Subsequently, a part of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, and 17.5 mL of the above standard solution were sequentially poured into volumetric flasks with a capacity of 25 mL. Each transfer was accompanied by dilution to attain a final concentration of 50.00, 100.01, 150.01, 200.02, 250.02, 300.03 and 350.03 µg/mL. Preparation of sample solution: For UV spectroscopy: Weigh accurately equivalent of 10,0 mg of powdered Rifaximin pills in a volumetric flask with a capacity of 100 mL, then mix with 50 mL of diluent and sonicate for 15 minutes while stirring intermittently. Lastly, diluents are used for volume makeup. Pass the 0.45µm membrane filter through. Using diluents, 5 mL of this solution further diluted to 25 mL with diluents and mix. Regarding the quantitative examination of riflaximin in commercial formulations, ten tablets of (each containing 550 mg of the drug) were meticulously weighed, and the average tablet's mass was measured and recorded. After that, these tablets were crushed using a mortar, powdered to a very fine consistency, and thoroughly mixed to ensure homogeneity. From this powder, an amount containing 50 mg equivalent of rifaximin tablet powder was weighed exactly and then transfer into a 50mL volumetric flask and add in 30mL of diluents. To ensure complete extraction of the drug, the solution underwent sonicated for 30 minutes final volume made up with diluents. Subsequently, for about 15 min, the solution was allowed to settle so that suspended insoluble components might precipitate. Thereafter, a 0.45 µm membrane filter was used to filter the sample to eliminate insoluble substances, resulting in the formation of the tablet sample solution. The tablet sample solution was prepared by further diluting 5 mL to a final volume of 25 mL with diluents. This prepared sample was subjected to quantitative analysis of chromatographic analysis. Spectrophotometric investigations were conducted employing a twin beam spectrophotometer, Shimadzu UV 1800, manufactured in Japan, managed by UV-Probe software. A 1.00 cm quartz cell was used to measure the absorbance’s of solutions in comparison to a blank sample. For the determination of rifaximin, Six concentrations of working standard solutions, ranging from 5 to 50 µg mL − 1 were prepared. These solutions were subjected to scanning on the spectrophotometer in wavelength across a range of 200–400 nm. Method Development: For spectrophotometric analysis, a thorough examination of the rifaximin spectral patterns in various solvents was conducted. Water and acetonitrile (1:1) ratio emerged as the optimal spectrophotometric analysis solvent, as it yielded the most favorable spectra. Moreover, given that at a wavelength of 296 nm, the maximum absorbance values of the standard solutions were noted. Absorbance measurements for both standard and sample solutions were carried out at this specific wavelength. Regarding chromatographic optimization, meticulous adjustments were made to various parameters. Variations in temperature, column types/sizes, and mobile phase combinations were explored to achieve optimal peak characteristics, including excellent peak shape, high theoretical plate number, low retention time frame, and minimal tailing factor. Method Validation 36 – 37 : The developed spectrophotometric and HPLC methods for quantifying Rifaximin in pharmaceutical formulations underwent validation following the guidelines outlined in the Q2 (R1) of the International Conference on Harmonisation (ICH). This validation process encompassed key parameters including selectivity, ruggedness, linearity, precision, sensitivity, and system suitability. Linearity: To assess the the HPLC method's linearity, 20 µL of six standard solutions a range of concentrations of 50.00–350.03 µg mL − 1 were injected in triplicate into the HPLC system over three separate days. With reference to each concentration, the peak areas were recorded. Subsequently, constructed calibration graphs by plotting peak areas on the y-axis against concentrations on the x-axis. Similarly, for the spectrophotometric method, the linearity was evaluated through calculating the absorbance values of six reference solutions with concentrations between 5.16 and 51.58 µgmL − 1 in triplicate using the spectrophotometer during the course of three separate days. Calibration graphs were generated with concentrations illustrated on the x-axis and spectrophotometric responses (absorbance values) on the y-axis based on the absorbance values obtained for each concentration, which were reported. The data obtained from both analytical methods were used in a regression analysis using the least squares method. The approaches' linearity was assessed based on parameters including the regression equations' slope, intercept, correlation coefficient, and absolute mean recovery value. Accuracy: The accuracy of the methods was assessed using the "standard addition method." This involved adding three different quantities of powder standards to three separate sample solutions, at rates equivalent to 80%, 100%, and 120% of the analyte content. Following a thorough mixing process, the analyte contents of the resultant solutions were determined by analysing them using the proposed procedures. Next, the recovery values for the additional standard quantities were calculated. For every concentration level, triplicate analyses were performed, and the additional standard amount's percentage recovery results were computed. Precision: Precision measurements, both intraday and interday, were used to evaluate the approaches' accuracy. The sample solution was quantitatively analysed six times on the same day (n = 6) for UV Spectroscopy (20 µg mL − 1 ) and HPLC 200 µg mL − 1 to assess the intraday precision. By examining the sample solution over the course of three consecutive days (n = 9), inter-day precision was assessed. Peak areas and retention times were measured in the HPLC method, and values of the relative standard deviation (RSD) were computed using these parameters. Simultaneously, absorbance values were measured using the spectrophotometric technique, and corresponding RSD values were computed. Robustness: Small, purposeful changes were made to specific method circumstances in order to assess the chromatographic method's robustness. These modifications included adjusting the mobile phase's flow rate by ± 0.1 mLmin − 1 , adjusting the organic solvent content by ± 10%, and adjusting the detection wavelength by ± 2 nm. After every alteration, the chromatographic system was filled with the standard solution (200 µg mL − 1 ), and the outcomes were contrasted with those that were produced using the initial chromatographic conditions. For every case, the system appropriateness criteria were noted. By analysing the reference solution in triplicate, the impacts of these changes were examined. Similarly, little modifications were made to the spectrophotometric method to assess its robustness. These alterations involved slight modifications to the differences in organic solvents and the detection wavelengths (298 and 294 nm) (switching between acetonitrile and water). Triplicate Analysis of the reference solution was carried out to investigate the effects of these changes, with the outcomes then contrasted with the ones acquired using the initial spectrophotometric setup. Limits of Quantification (LOQ) and Detection (LOD): To determine the Limits of quantification (LOQ) and detection (LOD) of the proposed methods, the standard deviation technique was employed. Blank samples, devoid of Rifaximin, were prepared for this purpose The chromatographic responses (peak areas) of the blank samples were recorded after the blank solution was injected into the apparatus three times using the HPLC procedure. Similar procedures applied to the spectrophotometric approach included triplicate examinations of the blank solution using the method and documentation of the spectrophotometric responses (absorbance values). The values of LOD and LOQ were then computed using the formulas LOD = 3.3 × SD/S and LOQ = 10 × SD/S, where S is the calibration curve's slope and SD is the peak area's standard deviation. Specificity: Determining whether the analyte could be sufficiently separated in the presence of additional excipients often found in dosage forms and looking into the possibility of interfering impurities were essential steps in evaluating the specificity of the techniques. In order to assess the specificity of the chromatographic procedure, the HPLC system was filled with mobile phase, standard, and sample solutions. After that, the sample solution's chromatogram and the standard solution's chromatogram were compared. Through this comparison, any interfering peaks that might have emerged during the analyte peak's retention period might be examined. A spectrophotometer was used to scan the spectra of the standard, sample, and solvent—water and acetonitrile (1:1) ratio—with a wavelength range of 200–400 nm in order to assess the specificity of the spectrophotometric approach. The spectra that were acquired were then compared in order to find any differences and check for the presence of interference bands. System suitability : Six times at short, regular intervals, the standard solution (200 µg mL − 1 ) was injected into the HPLC system to evaluate the system relevance of the HPLC method. Retention durations, peak sizes, theoretical plate numbers, and tailing factors of peaks containing rifaximin were carefully documented during this procedure. Relative standard deviation values for peak areas and retention times were then calculated.Likewise, absorbance values of the standard solution (20 µg mL-1) were determined at brief regular intervals to assess the spectrophotometric method's applicability for the system. The relative standard deviations of the absorbance values were then computed. Methods applied to commercial formulations: In order to conduct a quantitative analysis of rifaximin in commercial formulations, ten tablets containing 10 mg of rifaximin were precisely weighed, and the average tablet's mass was recorded. After that, the tablets were broken up into a fine powder in a sanitised mortar and blended well to guarantee consistency. The powdered tablet was weighed exactly and then dissolved in 100 millilitres of diluent. To ensure complete extraction of the drug, the solution underwent sonication for 30 minutes. Subsequently, filtered through 0.45µ nylon filter to remove insoluble substances, resulting in the formation of the stock solution sample. The preparation of the sample solution was done by withdrawing 5mL obtaining of the stock solution and its dilution to a final volume of 25 mL with ultra-pure water and acetonitrile (1:1). This prepared sample solution was then quantitatively analyzed using the techniques that were developed. Assessment of greenness profiles: How eco-friendly the developed methods are evaluated using the AGREE Analytical Greenness Metric software (version 0.5 beta), accessible at https://mostwiedzy.pl/wojciech-wojnowski,174235-1/AGREE . AGREE employs key principles to gauge the environmental friendliness of analytical methods 38 – 40 . The score for greenness displayed at the center of the chart, represents a benchmark value weighted average and is rounded to two decimal places. Scores range from 0.00 to 1.00, where a score below 0.50 signifies an undesirable method, 0.50–0.75 suggests acceptability, and a score exceeding 0.75 indicates a good level of greenness 41 – 43 . Results Selecting the wavelength of detection: Prepared standard solutions using HPLC garde water and acetonitrile in the ratio of 1:1, underwent scanning within the wavelength 200–400 nm in the spectrophotometer's range. The wavelength at which Rifaximin displayed maximum absorption was 296 nm. The overlay spectrum of standard solutions for rifaximin is shown in Fig. 1 . Method conditions: The separation procedure was aided by a Phenomenex C18 column (250 × 4.6 mm, 5 µm), with the column temperature being kept constant at 25°C. A 1:1 mixture of acetonitrile and water was used as the mobile phase. Isocratic elution was carried out at a flow rate of 1.0 mL min − 1 .Twenty microliters of both standard and sample solutions were introduced into the apparatus for examination. The wavelength at which rifaximin was detected was 296 nm. UV-Spectrophotometric method Parameters: HPLC garde water with acetonitrile served as the spectrophotometric analysis solvent due to the optimal spectra achieved under this condition. The values of absorbance of rifaximin 1:1 mixtures of acetonitrile and HPLC garde water ratio were directly measured using the spectrophotometer. Additionally, given that the reference solutions' maximum absorbance values were observed at a wavelength of 296 nm, At this particular wavelength, the standard and sample solutions were evaluated against HPLC water and acetonitrile (1:1) solutions. Results of method validation: Specificity: Chromatograms of the sample, mobile phase, and standard solutions were examined and contrasted in order to evaluate the specificity of the HPLC procedure. During the analyte peak's retention time, no interfering peak(s) were seen. The HPLC method's chromatograms, which include those of the mobile phase as the sample, blank, and standard solution, are displayed in Fig. 2 . The spectra of the ultra-pure water and acetonitrile (1:1) used as the Standard, sample, and solvent were analysed in order to evaluate the specificity of the spectrophotometric approach. To find any spectral bands interfering with the analyte spectrum, the three spectra were compared and examined. In all of the spectra, there were no bands that interfered with the rifaximin bands. Similar to this, Fig. 3 shows the spectra of the mobile phase as well as those of the blank, sample, and standard solution acquired using the spectrophotometric approach. System suitability test: Table 1. The physicochemical properties of rifaximin Table 2 provides a summary of the critical parameters that have been determined for system compliance tests. Notably, rifaximin showed consistently low variability in both peak areas and retention times, as well as outstanding peak symmetry. Additionally, the calibration curves' correlation coefficients were higher than 0.999, proving that the techniques were appropriate even for data with complicated matrices. The results of the system conformance tests validate that the developed methodologies are appropriate for quantifying rifaximin. RP-HPLC Method UV- Spectrophotometric method Sample Peak Area Time of Retention min Peak symmetry Theoretical plate Absorbance 1 12695417 18.25 1.35 3512 0.881 2 12697654 18.251 1.36 3521 0.882 3 12689987 18.24 1.35 3542 0.880 4 12698753 18.253 1.34 3511 0.885 5 12687651 18.231 1.35 3521 0.884 6 12687652 18.243 1.36 3546 0.883 Average 12692852 18.24 1.35 3578 0.883 S.D 4856 0.01 0.01 15.00 0.002 %RSD 0.04 0.05 0.56 0.42 0.21 Table No-2. System suitability Parameters Linearity: In the HPLC procedure, rifaximin's peak areas and retention periods were measured after standard solutions were injected into the system. For every concentration level, the average peak area and retention time were calculated. The peak area was then plotted versus the standard solution concentration on a calibration graph. In the meantime, the absorbance values of standard solutions were measured using the spectrophotometric method in comparison to an empty solution. Each concentration level's average absorbance value was determined, and a calibration graph showing absorbance values plotted against standard solution concentrations was created. Regression analysis was used to assess the analytical methodologies' linearity data. The regression equation's slope and intercept were found using linear regression analysis with the least squares approximation. Table 3 provides a summary of the linearity studies' findings. The calibration curve showed a positive linear relationship in the concentration range of 5.16–51.58 µg mL – 1 , which is significant. Table 3 Linearity data Measurement RP-HPLC UV-Spectrophotometer Range of Concentration µg/mL 50.00-350.03 5.16–51.58 Slope of Calibration curve 63754 1.00 Intercept of calibration curve 86041 0.03 coefficient of correlation < 0.999 < 0.999 Limit of detection (LOD) (µg/mL) 0.1 0.1 Limit of quantification(LOQ) (µg/mL) 0.3 0.3 Recovery % n = 3 99.62–99.64 99.60-99.92 Precision: Precision HPLC Spectrophotometer Retention time(RT) Peak area %Assay Absorbance %Assay Intraday Average 18.24 12660400 100.05 0.892 99.89 SD 0.01 25881.73 0.16 0.02 0.862 %RSD 0.05 0.20 0.16 1.71 0.86 Interday Average 18.24 12678330 100.76 0.89 99.75 SD 0.05 14771.60 0.19 0.01 0.278 %RSD 0.25 0.12 0.19 1.68 0.28 Table 4 Results of Precision data Accuracy: In order to evaluate the precision of the techniques, solutions were created by adding rifaximin standard at concentrations of 80%, 100%, and 120% to the sample solution (20 µg mL − 1 for UV spectroscopy and 250 µg mL − 1 for HPLC). The sample solution was then subjected to analysis utilizing the established methods. After that, the percentage recovery values of the standard amounts that were added to the sample solution were computed. For the HPLC method, the recovery percentages ranged from 99.62–99.64%, whereas for the spectrophotometric approach, they were between 99.62% and 99.92%. For the chromatographic method, the maximum relative standard deviation values were 0.49, while for the spectrophotometric method, they were 0.76. Table 5 illustrates the specific results from the recovery studies. Method Level in % Concentration in µgm/mL Mean Recovery % S.D %RSD HPLC method 80 40.14 99.62 0.49 0.49 100 49.92 99.66 0.04 0.04 120 59.83 99.64 0.51 0.51 UV spectroscopic method 80 8.38 99.92 0.60 0.60 100 10.18 99.60 0.76 0.76 120 12.28 99.92 0.58 0.58 Table 5. Results of Accuracy data Robustness: Little deviations from the norm were made in the method parameters, and the effects of these changes on the outcomes were investigated, in order to evaluate the robustness of the proposed approaches. This investigation was conducted for UV spectroscopy at a concentration level of 20 µg mL − 1 and for HPLC at a level of 250 µg mL − 1 .The greatest relative standard deviation value was found to be 0.89% based on the obtained results. Table 6 presents the results of the robustness tests as an overall result. Method Conditions Values Avg % assay % RSD HPLC Standard method 100.05 0.16 Plus Flow rate 1.10mL/min 100.21 0.36 Minus Flow rate 0.90mL/min 100.01 0.21 Plus Wavelength 298nm 100.39 0.45 Minus Wavelength 294 100.67 0.62 Plus Organic H2O:ACN(55:45) 100.23 0.32 Minus organic H2O:ACN(45:55) 100.12 0.21 UV- Spectroscopy Method Precision 99.89 0.86 Plus Wavelength 298nm 99.27 0.24 Minus Wavelength 294nm 99.80 0.89 Plus organic H2O:ACN(55:45) 100.34 0.60 Minus Organic H2O:ACN(45:55) 99.39 0.75 Table 6. Robustness data Rifaximin in marketed tablets: a quantitative analysis Quantitative analysis was performed on 6 tablets, each containing 550 mg of Rifaximin, Making use of the developed techniques. Table 7 summarises the analytical results along with the values of the standard deviation and relative standard deviation. Table 7 Results of Quantitative analysis of marketed tablets HPLC Spectrophotometer Sample Content in each tablet in Mg/WT % Assay Content in each tablet in Mg/WT % Assay 1 548.68 99.76 546.49 99.36 2 549.26 99.87 552.73 100.50 3 548.90 99.80 549.61 99.93 4 549.25 99.86 546.49 99.36 5 546.51 99.37 553.36 100.61 6 549.61 99.93 550.24 100.04 Average 548.704 99.76 549.822 99.97 S.D 1.12 0.19 2.94 0.49 %RSD 0.20 0.19 0.54 0.49 An evaluation of eco-friendly characteristics or sustainability profiles. The developed techniques' AGREE graphical representations are displayed on Fig. 4 . The developed HPLC and spectrophotometric techniques received AGREE analytical ratings of 0.80 and 0.90, respectively. A score of less than 0.50 on the AGREE greenness scale denotes insufficiency, a score between 0.50 and 0.75 is considered reasonable, and a score higher than 0.75 denotes excellent. Both of the suggested analytical techniques for rifaximin quantification are very ecologically friendly methodologies, according to the AGREE analytical evaluations obtained from this study 44 – 45 . The AGREE tool demonstrates the fulfillment of Principles of Green Chemistry. However, The use of energy is essential consideration, particularly in spectrophotometric and chromatographic techniques, where the effects on the environment can be quantified using the carbon footprint. The carbon footprint, measured in kg CO2 equivalent, assesses the adverse effects of a methodology, incorporating factors such as device power, duration of operation, and emission of electricity. According to Ballester-Caudet et al 2019 description, this metric is computed within the HEXAGON tool using the formula found in ESI S1 46 . The spectrophotometric method produced a total carbon footprint of 0.00550 kg CO2 equivalent, while the HPLC method produced 0.00765 kg CO2 equivalent according to the HEXAGON method. Based on the HEXAGON method, the ultimate score is 0 out of 5 points since both values are below 0.1. On a 5-point rating system, the overall competency score is zero for a carbon footprint that is less than 0.1. Our approaches' 5-minute analysis time is correlated with a lower carbon footprint score, making them more environmentally friendly 47 – 48 . Moreover, the HPLC approach results in a significant drop in overall expenses because less solvent is used in many tests as opposed to a single-component analysis. A number of variables, such as weekly sample throughput, equipment costs, and total analysis time, affect how cost-effective the procedure is. Evaluating fifty or more samples every week is said to be very eco-friendly. According to the HEXAGON tool's description, the procedure exhibits a high green value in terms of economic cost in our scenario, which involves analysing about 500 samples in seven days. Discussion This study used operator- and environmentally-friendly solvents to investigate the chromatographic and spectrophotometric behaviour of Rifaximin.From sample preparation to detection, the new methods' environmental friendliness was assessed. Sample preparation involved the use of only water, making the process simple and environmentally benign. In chromatographic analyses, water and acetonitrile, known for its safety for the health of analysts and the environment, was employed in the role of an organic modifier, while For spectrophotometric analyses, acetonitrile and ultrapure water were used as the solvents. The developed procedures performed exceptionally well after being validated in accordance with ICH recommendations our procedures showed excellent linearity, precision, accuracy, specificity, and robustness when used to the selective analysis of rifaximin. It was discovered that the developed procedures had extremely low detection and quantification limitations. In addition, the system suitability parameters demonstrated adequate method performance. This study's key finding is that the trash produced is non-toxic. The study's goals were accomplished when all validation requirements were satisfied without lowering performance standards. This research demonstrates the successful application of acetonitrile- and water-based mobile phases in pharmaceutical analyses, as well as the feasibility of conducting spectrophotometric analyses. Conclusions As clean water sources dwindle and air pollution escalates, the imperative to develop environmentally conscious methods becomes increasingly critical for humanity's future. Addressing environmental pollution, managing waste, and curbing energy consumption are paramount concerns. In this context, the development of eco-friendly methodologies assumes heightened significance. The suggested approach emerges as a more ecologically friendly sustainable alternative for quantifying rifaximin in pharmaceutical products compared to existing techniques. Leveraging highly eco-friendly solvents such as acetonitrile and water, the developed strategies provide a greener and user-friendly methodology for rifaximin analysis in pharmaceutical formulations. Effectively used to determine rifaximin in marketed dosage forms, these methods uphold environmental stewardship. Utilizing the AGREE assessment tool, the degree of greenness was verified, reaffirming the eco-friendly nature of the proposed method. Notably, the a survey of the literature showed There isn't a technique in use yet for determining Rifaximin that uses a mobile phase made of safer solvents The results of this investigation provide a basis for the shift from traditional chromatographic techniques to more ecologically friendly ones, all the while preserving acceptable levels of performance. Declarations Conflicting interests The writers say they have no conflicting motives. Author Contribution Authors Saroj Kanta Bisoyi carried out the research work and manuscript writing, Debasish Pradhan reviewed the data, Sudhir Kumar Sahoo and Umashankar Mishra carried out the statistical data analysis, Adyasa Samantaray and Bandana Behera prepared figures.All authors reviewed the manuscript. Acknowledgements: For supplying the resources necessary for this activity to be completed successfully. 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Development and Validation of Stability-Indicating Liquid Chromatographic Assay for Rifaximin (An Antibiotic) in Bulk and Pharmaceutical Dosage Forms. Forms Chem Sci Trans. 2014;3:566–75. Challa BR, Kotaiah MR, Chandu BR, Chandrasekhar KB, Kanchanamala K, Parveen SK, Micheal F. HPLC method for determination of rifaximin in human plasma using tandem mass spectrometry detection. East and Central African Journal of Pharmaceutical Sciences. 2010;13(3). Nageswara Rao R, Vali RM, Ramachandra B, Maurya PK. Rapid determination of rifaximin on dried blood spots by LC-ESI‐MS. Biomedical Chromatography. 2011;25(11):1201–7. Tzima K, Brunton NP, Rai DK. Qualitative and quantitative analysis of polyphenols in Lamiaceae plants—A review. Plants. 2018;7(2):25. Zhang X, Duan J, Li K, Zhou L, Zhai S. Sensitive quantification of rifaximin in human plasma by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B. 2007;850(1–2):348–55. Stradi R, Nava D, Nebuloni M, Pastura B, Pini E. Structural elucidation of the rifaximin Ph. Eur. impurity H. Journal of pharmaceutical and biomedical analysis. 2010;51(4):858–65. Rao RN, Vali RM, Shinde DD. On-line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum. Biomedical Chromatography. 2009;23(11):1145–50. Kogawa AC, Peltonen L, Salgado HR, Chorilli M. Short-Stability Study of Rifaximin-Based Samples. Journal of AOAC International. 2020;103(3):743–6. Kumar PR, Chary TJ, Sahithi K, Shareef AB, Babu NR. Study of Effect of Solvents on Absorption Characteristics of Rifaximin in Visible Region and Its Estimation in Bulk and Dosage Forms. Journal of Pharmaceutical Sciences and Research. 2019;11(5):2007–11. Masih AF. Forced degradation study of rifaximin formulated tablets to determine stability indicating nature of hplc method. International Journal of Chemical & Pharmaceutical Analysis. 2017;4(3). Viscomi GC, Campana M, Barbanti M, Grepioni F, Polito M, Confortini D, Rosini G, Righi P, Cannata V, Braga D. Crystal forms of rifaximin and their effect on pharmaceutical properties. CrystEngComm. 2008;10(8):1074–81. Boateng GO, Neilands TB, Frongillo EA, Melgar-Quiñonez HR, Young SL. Best practices for developing and validating scales for health, social, and behavioral research: a primer. Frontiers in public health. 2018;6:149. Ma L, Wang Z, Liu H, Wu C, Ding Y, Wen K. Monoclonal antibody production and the development of a quantitative time-resolved fluoroimmunoassay for rifaximin in milk. Food and agricultural immunology. 2019;30(1):1135–47. Žuvela P, Skoczylas M, Jay Liu J, Ba̧czek T, Kaliszan R, Wong MW, Buszewski B. Column characterization and selection systems in reversed-phase high-performance liquid chromatography. Chemical reviews. 2019;119(6):3674–729. Patyra E, Kwiatek K. Analytical capabilities of micellar liquid chromatography and application to residue and contaminant analysis: A review. Journal of Separation Science. 2021;44(11):2206–20. Kasimala BB, Kasimala MB. New spectrophotometric methods for the quantitative estimation of Rifaximin in formulations. Journal of Atoms and Molecules. 2011;1(1):48. Srinidhi B. S, Jose Gnana Babu C and Senthil Kumar G. P. RP-UFLC method development and validation of rifaximin in bulk and tablet dosage form. ssrg international journal of pharmacy and biomedical engineering, vol. 6, no. 1, pp. 35–39, 2019. Campanella B, Lomonaco T, Benedetti E, Onor M, Nieri R, Bramanti E. Validation and application of a derivatization-free RP-HPLC-DAD method for the determination of low molecular weight salivary metabolites. International Journal of Environmental Research and Public Health. 2020;17(17):6158. Demir İ, Bulduk İ, Darwısh IA, Enginar H. A green approach for metoclopramide quantification in pharmaceutical products: new HPLC and spectrophotometric methods. Scientific Reports. 2024;14(1):8765. Masih AF. Forced degradation study of rifaximin formulated tablets to determine stability indicating nature of HPLC method. International Journal of Chemical & Pharmaceutical Analysis. 2017;4(3). Pena-Pereira F, Wojnowski W, Tobiszewski M. AGREE—Analytical GREEnness metric approach and software. Analytical chemistry. 2020;92(14):10076–82. Constable DJ, Dunn PJ, Hayler JD, Humphrey GR, Leazer Jr JL, Linderman RJ, Lorenz K, Manley J, Pearlman BA, Wells A, Zaks A. Key green chemistry research areas—a perspective from pharmaceutical manufacturers. Green Chemistry. 2007;9(5):411–20. Polshettiwar V, Varma RS. Green chemistry by nano-catalysis. Green Chemistry. 2010;12(5):743–54. Jiménez-González C, Poechlauer P, Broxterman QB, Yang BS, Am Ende D, Baird J, Bertsch C, Hannah RE, Dell’Orco P, Noorman H, Yee S. Key green engineering research areas for sustainable manufacturing: a perspective from pharmaceutical and fine chemicals manufacturers. Organic Process Research & Development. 2011;15(4):900–11. Gałuszka A, Migaszewski ZM, Konieczka P, Namieśnik J. Analytical Eco-Scale for assessing the greenness of analytical procedures. TrAC Trends in Analytical Chemistry. 2012;37:61–72. Anastas PT, Bartlett LB, Kirchhoff MM, Williamson TC. The role of catalysis in the design, development, and implementation of green chemistry. Catalysis today. 2000;55(1–2):11–22. Alam P, Shakeel F, Alshehri S, Iqbal M, Foudah AI, Aljarba TM, Abdel Bar F, Alqarni MH. Simultaneous Estimation of Lesinurad and Allopurinol in the New FDA-Approved Products Using a Greener Normal-Phase HPTLC Method: Greenness Assessment Using the NEMI, Analytical Eco-Score, ChlorTox, and AGREE Methods. ACS Sustainable Chemistry & Engineering. 2024;12(4):1526–35. Huddleston JG, Visser AE, Reichert WM, Willauer HD, Broker GA, Rogers RD. Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation. Green chemistry. 2001;3(4):156–64. Ballester-Caudet A, Campíns-Falcó P, Pérez B, Sancho R, Lorente M, Sastre G, González C. A new tool for evaluating and/or selecting analytical methods: Summarizing the information in a hexagon. TrAC Trends in Analytical Chemistry. 2019; 118:538–47. Bang PP, Bhatt HG. Development of green RP-and green NP-HPTLC methods for estimation of lenvatinib and comparative evaluation by AGREE. ACS Sustainable Chemistry & Engineering. 2023;11(6):2249–63. Hammouda ME, Salem YA, El-Ashry SM, El-Enin MA. Inclusive study for sustainable enantioseparation of racemic chlorpheniramine and caffeine by HPLC using dual cyclodextrin system as chiral mobile phase additive: Assessment with AGREE and Complex-GAPI approaches. Sustainable Chemistry and Pharmacy. 2023; 35:101201. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4394184","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":309599143,"identity":"c0cc8a6a-1e5b-4481-acd1-943004ceb264","order_by":0,"name":"Saroj Kanta Bisoyi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYLACyQYJIMnY+CDBQEIOJHDgAZFamg0+FNgYg7UkENLC2ACm2CRnfEhLBLPxaZFvP/vwgeUOC3t+ieQGaR6Dw+nzww4/BNpiJ6fbgF2LwZl0YwPJMxKJM2ckNhgDteRuvJ1mANSSbGx2AIcWhjQ2Cck2iQSDG4kNyWAtsxNAWg4kbsOhRb7/GViLPUjLYZDDDGenf8CrheEGxBbGDTcSGxtnGKQlyEvn4LfF4MYzZohfeh42M3wwsDHcIJ1TcCDBALdf5PvTGB9L7qiz52dPf/4j4Y+EvPzs9M0fPlTYyeHSAgLMoIhkEEiA2nsAEix4AeMHEMkPNVS+Ab/qUTAKRsEoGHkAAGgEZAKdHHJaAAAAAElFTkSuQmCC","orcid":"","institution":"Royal College of Pharmacy and Health Sciences,Berhampur Odisha","correspondingAuthor":true,"prefix":"","firstName":"Saroj","middleName":"Kanta","lastName":"Bisoyi","suffix":""},{"id":309599144,"identity":"f28fcd6f-14a0-4e3a-9924-f1059a6a6268","order_by":1,"name":"Debasish Pradhan","email":"","orcid":"","institution":"Utkal University","correspondingAuthor":false,"prefix":"","firstName":"Debasish","middleName":"","lastName":"Pradhan","suffix":""},{"id":309599145,"identity":"c4ae766d-1711-47f5-acb1-66b9bd528fd5","order_by":2,"name":"sudhir Sahoo","email":"","orcid":"","institution":"Royal College of Pharmacy and Health Sciences,Berhampur Odisha","correspondingAuthor":false,"prefix":"","firstName":"sudhir","middleName":"","lastName":"Sahoo","suffix":""},{"id":309599146,"identity":"18d2b668-a984-49f9-a176-e38c0cf098b7","order_by":3,"name":"Umashankar Mishra","email":"","orcid":"","institution":"Royal College of Pharmacy and Health Sciences,Berhampur Odisha","correspondingAuthor":false,"prefix":"","firstName":"Umashankar","middleName":"","lastName":"Mishra","suffix":""},{"id":309599147,"identity":"702b82b3-c44e-40aa-b79f-d25fb09faddb","order_by":4,"name":"Adyasa Samantaray","email":"","orcid":"","institution":"Utkal University","correspondingAuthor":false,"prefix":"","firstName":"Adyasa","middleName":"","lastName":"Samantaray","suffix":""},{"id":309599148,"identity":"5b87d435-41a3-4959-ad88-eaac94fd92f4","order_by":5,"name":"Bandana Behera","email":"","orcid":"","institution":"C V Raman Global University Bhubaneswar","correspondingAuthor":false,"prefix":"","firstName":"Bandana","middleName":"","lastName":"Behera","suffix":""}],"badges":[],"createdAt":"2024-05-09 09:38:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4394184/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4394184/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57650393,"identity":"d0c4f2bf-2b18-4bf0-9e40-0245a650073f","added_by":"auto","created_at":"2024-06-03 22:22:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18656,"visible":true,"origin":"","legend":"\u003cp\u003eThe Rifaximin standard solutions' single spectrum in the 51.58 μg mL\u003csup\u003e−1\u003c/sup\u003e concentration range\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4394184/v1/988943401e376d59b86c9d39.png"},{"id":57650391,"identity":"f1162adc-4292-43cd-8118-1c1dadfb60ed","added_by":"auto","created_at":"2024-06-03 22:22:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":450257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecificity data\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4394184/v1/7902739684e4cc5dbfc3d7db.png"},{"id":57650390,"identity":"073b334e-2e80-46bb-addd-5a3ac881725f","added_by":"auto","created_at":"2024-06-03 22:22:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV spectra of Rifaximin\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4394184/v1/853f768439043a6b0a951bf3.png"},{"id":57650667,"identity":"ac01f022-46c2-487a-9a45-89136aa0f162","added_by":"auto","created_at":"2024-06-03 22:30:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130346,"visible":true,"origin":"","legend":"\u003cp\u003e(A) UV-Spectrometric method Agree pictograms. (B) Agree Pictogram scale (C) HPLC Method Agree pictogram\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4394184/v1/a013becdabe3db9c300c13df.png"},{"id":59404478,"identity":"60b85f9d-a202-4637-b2c8-19a14189933c","added_by":"auto","created_at":"2024-07-01 11:07:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2119073,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4394184/v1/b2a7f400-e8bc-4c5a-8a48-5a3ff8606f4b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel Green systematic HPLC and UV-Spectroscopic method for the determination of rifaximin in tablet formulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the last few years, there has been a notable surge in interest among researchers towards the advancement of green analytical methods\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This heightened attention stems from a collective effort to mitigate effects on the environment and safeguard the well-being of analysts\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Reverse phase liquid chromatography (RP-HPLC) stands as a pivotal technique in drug analysis within the pharmaceutical industry, playing a crucial role in both product development and the quality control test of pharmaceutical formulations \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTypically, HPLC techniques rely on a stationary phase that is hydrophobic in conjunction with the help of polar mobile phase to achieve effective separation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Commonly utilized column types such as C18 and C8 facilitate the separation of various components within the sample, thereby expediting HPLC studies, streamlining the analysis process, and ultimately reducing costs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Optimal column selection is imperative for ensuring satisfactory chromatographic peaks and achieving robust separation of analytes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn HPLC procedures, mobile phases are generally composed of a mixture of organic solvents such as acetonitrile and water, which is frequently enhanced with additives or buffer solutions to regulate pH. Because of its perfect miscibility with water, low viscosity, and limited chemical reactivity with device components and column surfaces, acetonitrile is a preferred option \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile factors like analysis time, accuracy, precision, and robustness have typically been given top priority in the development and validation of chromatographic methods, factors like the method\u0026apos;s influence on the environment and the safety of analysts have historically gained less attention. This underscores the need for a more comprehensive consideration of these factors in method development and validation processes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eModern antibiotics like rifaximin are essential for treating traveler\u0026apos;s diarrhoea in those 12 years of age and older, especially when non-invasive Escherichia coli strains are the cause. Derived from the antibiotic rifamycin, which is well-known for having strong antibacterial activity despite having a low rate of gastrointestinal absorption rifaximin works by attaching itself to the beta-subunit of RNA polymerase that is dependent on bacterial DNA. The catalytic step that is necessary to polymerize deoxyribonucleotides into a DNA strand is effectively hampered by this mechanism. Rifaximin Physiochemical Properties given in table no-1 \u003csup\u003e34\u0026ndash;35\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInstrument\u003c/h2\u003e \u003cp\u003eA double beam spectrophotometer, the Shimadzu UV 1700, was used for spectrophotometric investigations that was manufactured by Shimadzu, Japan, and was managed by UV-Probe software. The experiments were conducted within the UV region scanning with 200.0\u0026ndash;400.0 nm, with quartz cuvettes employed for sample containment.\u003c/p\u003e \u003cp\u003eFor HPLC analysis, an Agilent LC 1260 series system sourced from Agilent Technologies in the USA was utilized. Compound separation was accomplished with a Phenomenex C18 column (5.0 \u0026micro;m; 4.6 \u0026times; 250.0 mm in dimensions) purchased from Agilent in the United States.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMaterials:\u003c/h2\u003e \u003cp\u003eAll of the solvents used in liquid chromatography were gradiently pure. Rifaximin Active pharmaceutical ingredient as gift from pharmaceutical industries and Rifaximin tablets (550 mg) were acquired from a nearby drugstore for incorporation into this study.\u003c/p\u003e \u003cp\u003eTo prepare solutions along with the mobile phase, ultrapure water and acetonitrile was employed. Prior to analysis, the mobile phase underwent filtration using a 0.45 \u0026micro;m membrane filter under vacuum conditions and was subsequently sonicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of standard solutions:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eFor UV Spectroscopy\u003c/strong\u003e \u003cp\u003eWeigh accurately and transfer 10.34 mg of Rifaximin API into a volumetric flask with a capacity of 100 mL. To dissolve, sonicate the mixture after adding 50 millilitres of diluent. Add diluent to adjust the volume and stir. Further transfer 1.00, 2.00, 4.00, 6.00, 8.00 and 10.00 mL of standard stock transfer into 20 mL volumetric flask with volume added with diluents to attain the concentration of 5.16, 10.32, 20.63, 30.95, 41.26, 51.58 03 \u0026micro;g mL.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFor HPLC\u003c/b\u003e Accurately weigh and transfer 50.12 mg of Rifaximin API into a 100 mL volumetric flask. Add 50 mL of diluent to the mixture and sonicate to dissolve. To adjust the volume, add diluent and stir.\u003c/p\u003e \u003cp\u003eSubsequently, a part of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, and 17.5 mL of the above standard solution were sequentially poured into volumetric flasks with a capacity of 25 mL. Each transfer was accompanied by dilution to attain a final concentration of 50.00, 100.01, 150.01, 200.02, 250.02, 300.03 and 350.03 \u0026micro;g/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of sample solution:\u003c/h2\u003e \u003cp\u003eFor UV spectroscopy: Weigh accurately equivalent of 10,0 mg of powdered Rifaximin pills in a volumetric flask with a capacity of 100 mL, then mix with 50 mL of diluent and sonicate for 15 minutes while stirring intermittently. Lastly, diluents are used for volume makeup. Pass the 0.45\u0026micro;m membrane filter through. Using diluents, 5 mL of this solution further diluted to 25 mL with diluents and mix.\u003c/p\u003e \u003cp\u003eRegarding the quantitative examination of riflaximin in commercial formulations, ten tablets of (each containing 550 mg of the drug) were meticulously weighed, and the average tablet's mass was measured and recorded. After that, these tablets were crushed using a mortar, powdered to a very fine consistency, and thoroughly mixed to ensure homogeneity. From this powder, an amount containing 50 mg equivalent of rifaximin tablet powder was weighed exactly and then transfer into a 50mL volumetric flask and add in 30mL of diluents. To ensure complete extraction of the drug, the solution underwent sonicated for 30 minutes final volume made up with diluents. Subsequently, for about 15 min, the solution was allowed to settle so that suspended insoluble components might precipitate. Thereafter, a 0.45 \u0026micro;m membrane filter was used to filter the sample to eliminate insoluble substances, resulting in the formation of the tablet sample solution.\u003c/p\u003e \u003cp\u003eThe tablet sample solution was prepared by further diluting 5 mL to a final volume of 25 mL with diluents. This prepared sample was subjected to quantitative analysis of chromatographic analysis.\u003c/p\u003e \u003cp\u003eSpectrophotometric investigations were conducted employing a twin beam spectrophotometer, Shimadzu UV 1800, manufactured in Japan, managed by UV-Probe software. A 1.00 cm quartz cell was used to measure the absorbance\u0026rsquo;s of solutions in comparison to a blank sample.\u003c/p\u003e \u003cp\u003eFor the determination of rifaximin, Six concentrations of working standard solutions, ranging from 5 to 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were prepared. These solutions were subjected to scanning on the spectrophotometer in wavelength across a range of 200\u0026ndash;400 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMethod Development:\u003c/h2\u003e \u003cp\u003eFor spectrophotometric analysis, a thorough examination of the rifaximin spectral patterns in various solvents was conducted. Water and acetonitrile (1:1) ratio emerged as the optimal spectrophotometric analysis solvent, as it yielded the most favorable spectra. Moreover, given that at a wavelength of 296 nm, the maximum absorbance values of the standard solutions were noted. Absorbance measurements for both standard and sample solutions were carried out at this specific wavelength.\u003c/p\u003e \u003cp\u003eRegarding chromatographic optimization, meticulous adjustments were made to various parameters. Variations in temperature, column types/sizes, and mobile phase combinations were explored to achieve optimal peak characteristics, including excellent peak shape, high theoretical plate number, low retention time frame, and minimal tailing factor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethod Validation \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e:\u003c/h2\u003e \u003cp\u003eThe developed spectrophotometric and HPLC methods for quantifying Rifaximin in pharmaceutical formulations underwent validation following the guidelines outlined in the Q2 (R1) of the International Conference on Harmonisation (ICH). This validation process encompassed key parameters including selectivity, ruggedness, linearity, precision, sensitivity, and system suitability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLinearity:\u003c/h2\u003e \u003cp\u003eTo assess the the HPLC method's linearity, 20 \u0026micro;L of six standard solutions a range of concentrations of 50.00\u0026ndash;350.03 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were injected in triplicate into the HPLC system over three separate days. With reference to each concentration, the peak areas were recorded. Subsequently, constructed calibration graphs by plotting peak areas on the y-axis against concentrations on the x-axis.\u003c/p\u003e \u003cp\u003eSimilarly, for the spectrophotometric method, the linearity was evaluated through calculating the absorbance values of six reference solutions with concentrations between 5.16 and 51.58 \u0026micro;gmL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in triplicate using the spectrophotometer during the course of three separate days. Calibration graphs were generated with concentrations illustrated on the x-axis and spectrophotometric responses (absorbance values) on the y-axis based on the absorbance values obtained for each concentration, which were reported.\u003c/p\u003e \u003cp\u003eThe data obtained from both analytical methods were used in a regression analysis using the least squares method.\u003c/p\u003e \u003cp\u003eThe approaches' linearity was assessed based on parameters including the regression equations' slope, intercept, correlation coefficient, and absolute mean recovery value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAccuracy:\u003c/h2\u003e \u003cp\u003eThe accuracy of the methods was assessed using the \"standard addition method.\" This involved adding three different quantities of powder standards to three separate sample solutions, at rates equivalent to 80%, 100%, and 120% of the analyte content. Following a thorough mixing process, the analyte contents of the resultant solutions were determined by analysing them using the proposed procedures. Next, the recovery values for the additional standard quantities were calculated. For every concentration level, triplicate analyses were performed, and the additional standard amount's percentage recovery results were computed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrecision:\u003c/h2\u003e \u003cp\u003ePrecision measurements, both intraday and interday, were used to evaluate the approaches' accuracy. The sample solution was quantitatively analysed six times on the same day (n\u0026thinsp;=\u0026thinsp;6) for UV Spectroscopy (20 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and HPLC 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to assess the intraday precision. By examining the sample solution over the course of three consecutive days (n\u0026thinsp;=\u0026thinsp;9), inter-day precision was assessed.\u003c/p\u003e \u003cp\u003ePeak areas and retention times were measured in the HPLC method, and values of the relative standard deviation (RSD) were computed using these parameters. Simultaneously, absorbance values were measured using the spectrophotometric technique, and corresponding RSD values were computed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRobustness:\u003c/h2\u003e \u003cp\u003eSmall, purposeful changes were made to specific method circumstances in order to assess the chromatographic method's robustness. These modifications included adjusting the mobile phase's flow rate by \u0026plusmn;\u0026thinsp;0.1 mLmin\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, adjusting the organic solvent content by \u0026plusmn;\u0026thinsp;10%, and adjusting the detection wavelength by \u0026plusmn;\u0026thinsp;2 nm. After every alteration, the chromatographic system was filled with the standard solution (200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the outcomes were contrasted with those that were produced using the initial chromatographic conditions. For every case, the system appropriateness criteria were noted. By analysing the reference solution in triplicate, the impacts of these changes were examined.\u003c/p\u003e \u003cp\u003eSimilarly, little modifications were made to the spectrophotometric method to assess its robustness. These alterations involved slight modifications to the differences in organic solvents and the detection wavelengths (298 and 294 nm) (switching between acetonitrile and water). Triplicate Analysis of the reference solution was carried out to investigate the effects of these changes, with the outcomes then contrasted with the ones acquired using the initial spectrophotometric setup.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLimits of Quantification (LOQ) and Detection (LOD):\u003c/h2\u003e \u003cp\u003eTo determine the Limits of quantification (LOQ) and detection (LOD) of the proposed methods, the standard deviation technique was employed. Blank samples, devoid of Rifaximin, were prepared for this purpose The chromatographic responses (peak areas) of the blank samples were recorded after the blank solution was injected into the apparatus three times using the HPLC procedure. Similar procedures applied to the spectrophotometric approach included triplicate examinations of the blank solution using the method and documentation of the spectrophotometric responses (absorbance values). The values of LOD and LOQ were then computed using the formulas LOD\u0026thinsp;=\u0026thinsp;3.3 \u0026times; SD/S and LOQ\u0026thinsp;=\u0026thinsp;10 \u0026times; SD/S, where S is the calibration curve's slope and SD is the peak area's standard deviation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSpecificity:\u003c/h2\u003e \u003cp\u003eDetermining whether the analyte could be sufficiently separated in the presence of additional excipients often found in dosage forms and looking into the possibility of interfering impurities were essential steps in evaluating the specificity of the techniques.\u003c/p\u003e \u003cp\u003eIn order to assess the specificity of the chromatographic procedure, the HPLC system was filled with mobile phase, standard, and sample solutions. After that, the sample solution's chromatogram and the standard solution's chromatogram were compared. Through this comparison, any interfering peaks that might have emerged during the analyte peak's retention period might be examined.\u003c/p\u003e \u003cp\u003eA spectrophotometer was used to scan the spectra of the standard, sample, and solvent\u0026mdash;water and acetonitrile (1:1) ratio\u0026mdash;with a wavelength range of 200\u0026ndash;400 nm in order to assess the specificity of the spectrophotometric approach. The spectra that were acquired were then compared in order to find any differences and check for the presence of interference bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eSystem suitability\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eSix times at short, regular intervals, the standard solution (200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was injected into the HPLC system to evaluate the system relevance of the HPLC method. Retention durations, peak sizes, theoretical plate numbers, and tailing factors of peaks containing rifaximin were carefully documented during this procedure. Relative standard deviation values for peak areas and retention times were then calculated.Likewise, absorbance values of the standard solution (20 \u0026micro;g mL-1) were determined at brief regular intervals to assess the spectrophotometric method's applicability for the system. The relative standard deviations of the absorbance values were then computed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMethods applied to commercial formulations:\u003c/h2\u003e \u003cp\u003eIn order to conduct a quantitative analysis of rifaximin in commercial formulations, ten tablets containing 10 mg of rifaximin were precisely weighed, and the average tablet's mass was recorded. After that, the tablets were broken up into a fine powder in a sanitised mortar and blended well to guarantee consistency. The powdered tablet was weighed exactly and then dissolved in 100 millilitres of diluent. To ensure complete extraction of the drug, the solution underwent sonication for 30 minutes. Subsequently, filtered through 0.45\u0026micro; nylon filter to remove insoluble substances, resulting in the formation of the stock solution sample.\u003c/p\u003e \u003cp\u003eThe preparation of the sample solution was done by withdrawing 5mL obtaining of the stock solution and its dilution to a final volume of 25 mL with ultra-pure water and acetonitrile (1:1). This prepared sample solution was then quantitatively analyzed using the techniques that were developed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of greenness profiles:\u003c/h2\u003e \u003cp\u003eHow eco-friendly the developed methods are evaluated using the AGREE Analytical Greenness Metric software (version 0.5 beta), accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mostwiedzy.pl/wojciech-wojnowski,174235-1/AGREE\u003c/span\u003e\u003cspan address=\"https://mostwiedzy.pl/wojciech-wojnowski,174235-1/AGREE\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. AGREE employs key principles to gauge the environmental friendliness of analytical methods \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The score for greenness displayed at the center of the chart, represents a benchmark value weighted average and is rounded to two decimal places. Scores range from 0.00 to 1.00, where a score below 0.50 signifies an undesirable method, 0.50\u0026ndash;0.75 suggests acceptability, and a score exceeding 0.75 indicates a good level of greenness \u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eSelecting the wavelength of detection:\u003c/h2\u003e\n \u003cp\u003ePrepared standard solutions using HPLC garde water and acetonitrile in the ratio of 1:1, underwent scanning within the wavelength 200\u0026ndash;400 nm in the spectrophotometer\u0026apos;s range. The wavelength at which Rifaximin displayed maximum absorption was 296 nm. The overlay spectrum of standard solutions for rifaximin is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eMethod conditions:\u003c/h2\u003e\n \u003cp\u003eThe separation procedure was aided by a Phenomenex C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m), with the column temperature being kept constant at 25\u0026deg;C. A 1:1 mixture of acetonitrile and water was used as the mobile phase. Isocratic elution was carried out at a flow rate of 1.0 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.Twenty microliters of both standard and sample solutions were introduced into the apparatus for examination. The wavelength at which rifaximin was detected was 296 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eUV-Spectrophotometric method Parameters:\u003c/h2\u003e\n \u003cp\u003eHPLC garde water with acetonitrile served as the spectrophotometric analysis solvent due to the optimal spectra achieved under this condition. The values of absorbance of rifaximin 1:1 mixtures of acetonitrile and HPLC garde water ratio were directly measured using the spectrophotometer. Additionally, given that the reference solutions\u0026apos; maximum absorbance values were observed at a wavelength of 296 nm, At this particular wavelength, the standard and sample solutions were evaluated against HPLC water and acetonitrile (1:1) solutions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eResults of method validation:\u003c/h2\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eSpecificity:\u003c/h2\u003e\n \u003cp\u003eChromatograms of the sample, mobile phase, and standard solutions were examined and contrasted in order to evaluate the specificity of the HPLC procedure. During the analyte peak\u0026apos;s retention time, no interfering peak(s) were seen. The HPLC method\u0026apos;s chromatograms, which include those of the mobile phase as the sample, blank, and standard solution, are displayed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe spectra of the ultra-pure water and acetonitrile (1:1) used as the Standard, sample, and solvent were analysed in order to evaluate the specificity of the spectrophotometric approach. To find any spectral bands interfering with the analyte spectrum, the three spectra were compared and examined. In all of the spectra, there were no bands that interfered with the rifaximin bands. Similar to this, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the spectra of the mobile phase as well as those of the blank, sample, and standard solution acquired using the spectrophotometric approach.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eSystem suitability test:\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe physicochemical properties of rifaximin\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1717453072.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eprovides a summary of the critical parameters that have been determined for system compliance tests. Notably, rifaximin showed consistently low variability in both peak areas and retention times, as well as outstanding peak symmetry. Additionally, the calibration curves\u0026apos; correlation coefficients were higher than 0.999, proving that the techniques were appropriate even for data with complicated matrices. The results of the system conformance tests validate that the developed methodologies are appropriate for quantifying rifaximin.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eRP-HPLC Method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUV- Spectrophotometric method\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak Area\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime of Retention min\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak symmetry\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTheoretical plate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbsorbance\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e12695417\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e18.25\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e3512\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.881\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12697654\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.251\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3521\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.882\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12689987\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3542\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.880\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12698753\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.253\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3511\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.885\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12687651\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.231\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3521\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.884\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12687652\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.243\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3546\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.883\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12692852\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3578\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.883\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eS.D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4856\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.56\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\u003cstrong\u003eTable No-2. System suitability Parameters\u003c/strong\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eLinearity:\u003c/h2\u003e\n \u003cp\u003eIn the HPLC procedure, rifaximin\u0026apos;s peak areas and retention periods were measured after standard solutions were injected into the system. For every concentration level, the average peak area and retention time were calculated. The peak area was then plotted versus the standard solution concentration on a calibration graph. In the meantime, the absorbance values of standard solutions were measured using the spectrophotometric method in comparison to an empty solution. Each concentration level\u0026apos;s average absorbance value was determined, and a calibration graph showing absorbance values plotted against standard solution concentrations was created.\u003c/p\u003e\n \u003cp\u003eRegression analysis was used to assess the analytical methodologies\u0026apos; linearity data. The regression equation\u0026apos;s slope and intercept were found using linear regression analysis with the least squares approximation. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e provides a summary of the linearity studies\u0026apos; findings. The calibration curve showed a positive linear relationship in the concentration range of 5.16\u0026ndash;51.58 \u0026micro;g mL\u003csup\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, which is significant.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLinearity data\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMeasurement\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRP-HPLC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUV-Spectrophotometer\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRange of Concentration \u0026micro;g/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e50.00-350.03\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5.16\u0026ndash;51.58\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSlope of Calibration curve\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e63754\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntercept of calibration curve\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e86041\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecoefficient of correlation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.999\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.999\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLimit of detection (LOD) (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLimit of quantification(LOQ) (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery % n\u0026thinsp;=\u0026thinsp;3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.62\u0026ndash;99.64\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.60-99.92\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003ePrecision:\u003c/h2\u003e\n \u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.93718166383701%\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.38879456706282%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPLC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.67402376910017%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpectrophotometer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.454545454545453%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention time(RT)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.2020202020202%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%Assay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsorbance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.91919191919192%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%Assay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.389830508474576%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntraday\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.491525423728813%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.728813559322035%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.559322033898304%\"\u003e\n \u003cp\u003e\u003cstrong\u003e12660400\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.203389830508474%\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.271186440677965%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.892\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.35593220338983%\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.504892367906066%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.851272015655578%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.655577299412915%\"\u003e\n \u003cp\u003e\u003cstrong\u003e25881.73\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.090019569471623%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.786692759295498%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.111545988258317%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.862\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.504892367906066%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.851272015655578%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.655577299412915%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.090019569471623%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.786692759295498%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.71\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.111545988258317%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.86\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.389830508474576%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterday\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.491525423728813%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.728813559322035%\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.559322033898304%\"\u003e\n \u003cp\u003e\u003cstrong\u003e12678330\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.203389830508474%\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.76\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.271186440677965%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.35593220338983%\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.75\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.504892367906066%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.851272015655578%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.655577299412915%\"\u003e\n \u003cp\u003e\u003cstrong\u003e14771.60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.090019569471623%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.786692759295498%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.111545988258317%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.278\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.504892367906066%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.851272015655578%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.655577299412915%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.090019569471623%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.786692759295498%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.111545988258317%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cstrong\u003eResults of Precision data\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eAccuracy:\u003c/h2\u003e\n \u003cp\u003eIn order to evaluate the precision of the techniques, solutions were created by adding rifaximin standard at concentrations of 80%, 100%, and 120% to the sample solution (20 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for UV spectroscopy and 250 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for HPLC). The sample solution was then subjected to analysis utilizing the established methods. After that, the percentage recovery values of the standard amounts that were added to the sample solution were computed.\u003c/p\u003e\n \u003cp\u003eFor the HPLC method, the recovery percentages ranged from 99.62\u0026ndash;99.64%, whereas for the spectrophotometric approach, they were between 99.62% and 99.92%. For the chromatographic method, the maximum relative standard deviation values were 0.49, while for the spectrophotometric method, they were 0.76. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the specific results from the recovery studies.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLevel in %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration in \u0026micro;gm/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Recovery %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS.D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPLC method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e40.14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.62\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e49.92\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.66\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e120\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e59.83\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.64\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eUV spectroscopic method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.92\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.76\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.76\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e120\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e12.28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.92\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cstrong\u003eTable 5. Results of Accuracy data\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eRobustness:\u003c/h2\u003e\n \u003cp\u003eLittle deviations from the norm were made in the method parameters, and the effects of these changes on the outcomes were investigated, in order to evaluate the robustness of the proposed approaches. This investigation was conducted for UV spectroscopy at a concentration level of 20 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and for HPLC at a level of 250 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.The greatest relative standard deviation value was found to be 0.89% based on the obtained results. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e presents the results of the robustness tests as an overall result.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.4576802507837%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.25078369905956%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.15987460815047%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eValues\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAvg \u0026nbsp;% assay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.949843260188088%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e% \u0026nbsp;RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.4576802507837%\" rowspan=\"7\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPLC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.25078369905956%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.15987460815047%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.949843260188088%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlus Flow rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.10mL/min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinus Flow rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.90mL/min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlus Wavelength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e298nm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.39\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinus Wavelength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e294\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.62\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlus Organic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH2O:ACN(55:45)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinus organic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH2O:ACN(45:55)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.4576802507837%\" rowspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eUV- Spectroscopy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.25078369905956%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethod Precision\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.15987460815047%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.949843260188088%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.86\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlus Wavelength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e298nm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinus Wavelength\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e294nm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlus organic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH2O:ACN(55:45)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"36.210131332082554%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinus Organic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.328330206378986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH2O:ACN(45:55)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.76360225140713%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.39\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.69793621013133%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.75\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cstrong\u003eTable 6. Robustness data\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003eRifaximin in marketed tablets: a quantitative analysis\u003c/h2\u003e\n \u003cp\u003eQuantitative analysis was performed on 6 tablets, each containing 550 mg of Rifaximin, Making use of the developed techniques. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e summarises the analytical results along with the values of the standard deviation and relative standard deviation.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of Quantitative analysis of marketed tablets\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eHPLC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSpectrophotometer\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eContent in each tablet in Mg/WT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Assay\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eContent in each tablet in Mg/WT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Assay\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e548.68\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e99.76\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e546.49\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e99.36\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e549.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.87\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e552.73\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e548.90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e549.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.93\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e549.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.86\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e546.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e546.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.37\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e553.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e549.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.93\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e550.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e100.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e548.704\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.76\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e549.822\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eS.D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.94\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.54\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eAn evaluation of eco-friendly characteristics or sustainability profiles.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe developed techniques\u0026apos; AGREE graphical representations are displayed on Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The developed HPLC and spectrophotometric techniques received AGREE analytical ratings of 0.80 and 0.90, respectively. A score of less than 0.50 on the AGREE greenness scale denotes insufficiency, a score between 0.50 and 0.75 is considered reasonable, and a score higher than 0.75 denotes excellent. Both of the suggested analytical techniques for rifaximin quantification are very ecologically friendly methodologies, according to the AGREE analytical evaluations obtained from this study \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe AGREE tool demonstrates the fulfillment of Principles of Green Chemistry. However, The use of energy is essential consideration, particularly in spectrophotometric and chromatographic techniques, where the effects on the environment can be quantified using the carbon footprint. The carbon footprint, measured in kg CO2 equivalent, assesses the adverse effects of a methodology, incorporating factors such as device power, duration of operation, and emission of electricity. According to Ballester-Caudet et al 2019 description, this metric is computed within the HEXAGON tool using the formula found in ESI S1 \u003csup\u003e46\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe spectrophotometric method produced a total carbon footprint of 0.00550 kg CO2 equivalent, while the HPLC method produced 0.00765 kg CO2 equivalent according to the HEXAGON method. Based on the HEXAGON method, the ultimate score is 0 out of 5 points since both values are below 0.1.\u003c/p\u003e\n \u003cp\u003eOn a 5-point rating system, the overall competency score is zero for a carbon footprint that is less than 0.1. Our approaches\u0026apos; 5-minute analysis time is correlated with a lower carbon footprint score, making them more environmentally friendly \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eMoreover, the HPLC approach results in a significant drop in overall expenses because less solvent is used in many tests as opposed to a single-component analysis. A number of variables, such as weekly sample throughput, equipment costs, and total analysis time, affect how cost-effective the procedure is. Evaluating fifty or more samples every week is said to be very eco-friendly. According to the HEXAGON tool\u0026apos;s description, the procedure exhibits a high green value in terms of economic cost in our scenario, which involves analysing about 500 samples in seven days.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study used operator- and environmentally-friendly solvents to investigate the chromatographic and spectrophotometric behaviour of Rifaximin.From sample preparation to detection, the new methods' environmental friendliness was assessed.\u003c/p\u003e \u003cp\u003eSample preparation involved the use of only water, making the process simple and environmentally benign. In chromatographic analyses, water and acetonitrile, known for its safety for the health of analysts and the environment, was employed in the role of an organic modifier, while For spectrophotometric analyses, acetonitrile and ultrapure water were used as the solvents.\u003c/p\u003e \u003cp\u003eThe developed procedures performed exceptionally well after being validated in accordance with ICH recommendations our procedures showed excellent linearity, precision, accuracy, specificity, and robustness when used to the selective analysis of rifaximin. It was discovered that the developed procedures had extremely low detection and quantification limitations.\u003c/p\u003e \u003cp\u003eIn addition, the system suitability parameters demonstrated adequate method performance. This study's key finding is that the trash produced is non-toxic. The study's goals were accomplished when all validation requirements were satisfied without lowering performance standards.\u003c/p\u003e \u003cp\u003eThis research demonstrates the successful application of acetonitrile- and water-based mobile phases in pharmaceutical analyses, as well as the feasibility of conducting spectrophotometric analyses.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAs clean water sources dwindle and air pollution escalates, the imperative to develop environmentally conscious methods becomes increasingly critical for humanity's future. Addressing environmental pollution, managing waste, and curbing energy consumption are paramount concerns. In this context, the development of eco-friendly methodologies assumes heightened significance.\u003c/p\u003e \u003cp\u003eThe suggested approach emerges as a more ecologically friendly sustainable alternative for quantifying rifaximin in pharmaceutical products compared to existing techniques. Leveraging highly eco-friendly solvents such as acetonitrile and water, the developed strategies provide a greener and user-friendly methodology for rifaximin analysis in pharmaceutical formulations. Effectively used to determine rifaximin in marketed dosage forms, these methods uphold environmental stewardship.\u003c/p\u003e \u003cp\u003eUtilizing the AGREE assessment tool, the degree of greenness was verified, reaffirming the eco-friendly nature of the proposed method. Notably, the a survey of the literature showed There isn't a technique in use yet for determining Rifaximin that uses a mobile phase made of safer solvents\u003c/p\u003e \u003cp\u003eThe results of this investigation provide a basis for the shift from traditional chromatographic techniques to more ecologically friendly ones, all the while preserving acceptable levels of performance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicting interests\u003c/h2\u003e \u003cp\u003eThe writers say they have no conflicting motives.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors Saroj Kanta Bisoyi carried out the research work and manuscript writing, Debasish Pradhan reviewed the data, Sudhir Kumar Sahoo and Umashankar Mishra carried out the statistical data analysis, Adyasa Samantaray and Bandana Behera prepared figures.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eFor supplying the resources necessary for this activity to be completed successfully. The writers would like to express their gratitude to the Royal College of Pharmacy and Health Sciences in Berhampur.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAvailability of materials and data from the corresponding author whenever required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnastas P, Eghbali N. Green chemistry: principles and practice. Chemical Society Reviews. 2010;39(1):301\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalem H, Abdelmajed MA, Rabiey M, Saied O, Amir M, Abdelgalil M. Utility of green chemistry for sustainable fluorescence derivatization approach for spectrofluorimetric quantification of Darolutamide as antineoplastic drug in pharmaceutical formulation and spiked human plasma. Luminescence. 2024;39(3):e4704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAboelghar SM, Hegazy MA, Wagdy HA. 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Catalysis today. 2000;55(1\u0026ndash;2):11\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlam P, Shakeel F, Alshehri S, Iqbal M, Foudah AI, Aljarba TM, Abdel Bar F, Alqarni MH. Simultaneous Estimation of Lesinurad and Allopurinol in the New FDA-Approved Products Using a Greener Normal-Phase HPTLC Method: Greenness Assessment Using the NEMI, Analytical Eco-Score, ChlorTox, and AGREE Methods. ACS Sustainable Chemistry \u0026amp; Engineering. 2024;12(4):1526\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuddleston JG, Visser AE, Reichert WM, Willauer HD, Broker GA, Rogers RD. Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation. Green chemistry. 2001;3(4):156\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallester-Caudet A, Camp\u0026iacute;ns-Falc\u0026oacute; P, P\u0026eacute;rez B, Sancho R, Lorente M, Sastre G, Gonz\u0026aacute;lez C. A new tool for evaluating and/or selecting analytical methods: Summarizing the information in a hexagon. TrAC Trends in Analytical Chemistry. 2019; 118:538\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBang PP, Bhatt HG. Development of green RP-and green NP-HPTLC methods for estimation of lenvatinib and comparative evaluation by AGREE. ACS Sustainable Chemistry \u0026amp; Engineering. 2023;11(6):2249\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHammouda ME, Salem YA, El-Ashry SM, El-Enin MA. Inclusive study for sustainable enantioseparation of racemic chlorpheniramine and caffeine by HPLC using dual cyclodextrin system as chiral mobile phase additive: Assessment with AGREE and Complex-GAPI approaches. Sustainable Chemistry and Pharmacy. 2023; 35:101201.\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":"Rifaximin, tablet, eco-friendly RP-HPLC, environmentally conscious UV-spectrophotometer, Software AGREE assessment","lastPublishedDoi":"10.21203/rs.3.rs-4394184/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4394184/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA sustainable approach has been devised for quantifying rifaximin in pharmaceutical products, utilizing both spectrophotometric and HPLC methods. In the spectrophotometric technique, accurate measurement of absorbance at 296 nm detection wavelength was conducted by taking water and acetonitrile (1:1) as the solvent. For the HPLC method, a phenomenonex C18 250 x 4.6mm,5\u0026micro; column was employed, with water and acetonitrile (1:1 v/v) serving as the mobile phase. Isocratic separation elution technique was employed at a rate of 1 mL per minute, with detection of rifaximin at 296 nm. These methods were found to be cost-effective, quick, eco-friendly, and straight forward, giving an analysis of rifaximin in 20 minutes. Application of these methods to pharmaceutical products yielded results free from matrix interference, with statistical comparison showing no significant differences among the techniques. Furthermore, the assessment of greenness conducted using AGREE software highlighted the developed methods' is environmental friendliness, which rely on solvents such as acetonitrile and water. These findings advocate for the adoption of our both analyst- and environmentally-friendly methods for the determination of rifaximin in tablet formulation over presently employed techniques.\u003c/p\u003e","manuscriptTitle":"A novel Green systematic HPLC and UV-Spectroscopic method for the determination of rifaximin in tablet formulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 22:22:39","doi":"10.21203/rs.3.rs-4394184/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":"628c3789-49f5-4a64-bed8-6f32004393ef","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-01T10:59:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-03 22:22:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4394184","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4394184","identity":"rs-4394184","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}