A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector

A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector Chetan Chaudhary, Yamini Sharma, Abdul Shakir, S. K. Luthra, Ranjan Mitra, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7113859/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 Cocamidopropyl betaine (CAPB) is a moderate zwitterionic (amphoteric) synthetic surfactants commonly used in shampoos and other personal care products. They serve as key ingredients that help to cleanse, emulsify, and create foam, removing dirt, impurities from the skin and hair. CAPB has been increasingly used in personal care formulations because of its foam boosting and high mildness properties. This study aimed to develop and validate method for the quantification of CAPB in shampoo using HPLC-RI detector. All validation parameters were conducted in compliance with the ICH guidelines and AOAC International Appendix K and were found to be within permissible limits. R 2 for linearity was greater than 0.99. LOD and LOQ were 60 µg/mL and 200 µg/mL respectively. The % RSD for method precision was 0.85%. The recovery range was found within 95–98%. The developed method is precise, robust, independent of the Shampoo matrix and thus suitable for routine for CAPB identification and quantification in Shampoo. Shampoo Cocamidopropyl betaine (CAPB) Validation HPLC RI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction 1.1 Introduction Surfactants are a key group of compounds used in personal care products and detergents designed for both commercial and household applications [ 1 , 2 ]. They have both hydrophilic and hydrophobic heads that reduce the surface tension between liquids-liquid/ liquid-solid/gas-liquid [ 3 ]. Surfactants are categorized on their head charge nature i.e. anionic, cationic, non-ionic and Zwitterionic surfactants [ 3 ]. Isoelectric surfactants are molecules which have two ionic heads of opposite charge in single molecule [ 4 ]. Majority of the Zwitterionic surfactant are subtle to the human skin, which exhibits less lethal as within the same compound they have both negatively and positively charged structure and ensures cationic or anionic activity occurs within narrow pH range, avoiding both extremes [ 5 , 6 , 7 ]. Various surfactants which have different heads are used simultaneously to improve product performance. Cocamidopropyl betaine (CAPB) is a moderate zwitterionic (amphoteric) ammonium-containing synthetic surfactant that is manufactured extensively and used in related industries products such as shampoos, contact lens solutions, detergents, skin care products, cleansers, liquid soap and antiseptics [ 8 ]. Johnson & Johnson was first company to introduce Cocamidopropyl betaine (CAPB) into personal care products in 1967 as "No More Tears" ingredient in children's shampoos. [ 9 ]. As consumption continues to rise in personal care products like face wash, shampoo, etc. Natural products have struggled to keep up with their ongoing demand. Synthetic CAPB is one of the most widely used surfactant derivates derived from coconut oil commercially Cocamidopropyl betaine is frequently used in the manufacturing industry to cover the increasing demand for these products. The use of synthetic Cocamidopropyl betaine in personal care products can help to manage the rising demand and scarcity of natural resources. Coconut oil serves as a primary source of various types of fatty acids. [ 10 ]. Coconut oil is mixture of fatty acids with diverse chain lengths and contains specific types of fatty acids, i.e.: lauric acid, myristic acid, caprylic acid and capric acid [ 11 , 12 ]. The fatty acids derived from coconut oil, either as a mixture or individually isolated, can be chemically modified with water loving substances to create surfactants. CAPB is produced by reacting fatty acids, frequently sourced from coconut oil or coconut with dimethylaminopropylamine (DMAPA), resulting lauramindopropyldimethlamine (LAPDMA) [ 13 ]. LAPDMA is consequently permitted to react with sodium monochloroaceate to give the product CAPB. CAPB has an anionic part (carboxylic group) and cationic part (quaternary ammonium group). The fat-loving group is formed by a combination of fatty acids of coconut with chain lengths between C-8 and C-18. This indicates that CAPB consists of compounds that share a common basic structure but differ in their lipophilic. Free fatty acids and sterols are contained by other substances, which are commercial products. CAPB is typically supplied as a moderately viscous solution, with concentrations typically ranging between 25%w/w to 30% w/w for personal care products. CAPB concentration is typically denoted by its activity level. It is mainly calculated as the total percentage of solids subtracted by the percentage of NaCl [ 14 ]. Mixed surfactant system performs better than single surfactant system. Advance liquid detergents, personal care products are muti surfactant products with several interactions within the constituents that boost product effectiveness. Anionic and Amphoteric surfactants are basic surfactant used in personal care products to achieve outstanding detergency, cleansing quality, good foaming property and easy thickening in the presence of salt. CAPB has been the most important secondary surfactant for personal- cleansing products for a long time owing to the low irritation potential of the pure surfactant and its good toxicological properties with another anionic surfactant to reduces irritation to skin and mucus membrane and leads to a pleasant foam. CAPB is known to give a better cleansing performance and to improve the conditioning properties of hair. CAPB demand is increasing in personal care industries. Thus, accurately determining CAPB is crucial. In the past, various paper was published dealing with the solution of this problem using HPLC on different types of column and different detection techniques [ 15 ]. For surfactants, numerous analytical methods have been developed, including thin layer chromatography [ 16 ], titration with two-phase [ 17 , 18 ], HPLC on a cation exchange column [ 19 ], Prep-HPLC [ 20 ], Potentiometric analysis [ 21 ], UHPLC–MS/MS [ 21 ], ATR-FTIR[ 22 ]. These all-reported methods are highly dependent upon the matrix of the sample, or they required special type of column or sample pretreatment which require more time and large amount. If we talk about detectors like MS/MS, ATR_FTIR which are highly sophisticated detectors require high maintenance and high cost of analysis. Our method is substitute to these above methods. As far as we are aware, no method has been available based on HPLC coupled with a Refractive index on this column for the determination of CAPB in Shampoo. As RI detector effectively measures changes in the refractive index caused by the presence of CAPB in the sample, allowing accurate detection. RI detector maintenance is low, and cost of analysis is also cheap in comparison to MS and other detector. The column used is the simplest C18 column, which is readily available, cost effective, and does not require post- column treatment. The present study aims to develop and validate a new method based on HPLC-RI for exact determination of CAPB in Shampoo. The procedure underwent validation according to ICH guideline Q2 (R1). 2. Experimental 2.1 Materials and Reagents Reference standard of Cocamidopropyl betaine (Lot no. 2-MJJ-54-1, Purity-35.7%w/w)) purchased from Toronto Research Chemical Canada, Acetonitrile purchased from Merck Limited (Lot no. DE4DF74840, Merck Life Science Pvt. Ltd. Mumbai, India) 0.45 µ m PTFE syringe filter was procured from AXIVA (Lot no. SF0030119Axiva Sichem Pvt. Ltd. Sonipat, Haryana-131028, India). Water used was milli-Q-Water. Dabur vatika shampoo was taken for the analysis. 2.2 Instrumentation and Equipment Table 1 Instrument and equipment details Instrument/Equipment Name Make/Model Analytical Balance XS105 dual range balance (Mettler Toledo) Multi Tube Vortex Mixture (iSwix MV) Neuation Technologies Pvt. Ltd, 194/6/2, Dantali Industrial Estate, Gota -Vadsar Road Dantali, Kalol, Gandhinagar, Gujarat 382721Speed: 2500 RPM Eppendorf Centrifuge (5804 R) Eppendorf India Private Limited, Plot No. 18,19, 20-(Part) South Phase, SIDCO Industrial Estate, Amb attur, Chennai, Tamil Nadu, India, Max Speed: 14000 rpm HPLC 1.Shimadzu, Model LC-2030C 3D, with refractive index detector model no. 20A, Made in Japan 2.Agilent Technologies, model 1200, with refractive index detector model no. 1260 infinity II, Made in Germany Column HPLC ODS-3V Inertsil Column, (250 x 4.6 mm x 5µm) Catalogue. Number: 5020 − 01732, Serial Number: 20G0133298, made in Japan Volumetric flask 25 mL class A(Borosil) Centrifuge tube 50 mL (Tarson) 2.3 Chromatographic Condition Table 2 HPLC Chromatographic Condition details Detector Refractive index detector (RID) Flow Rate 1.0 ml/min MobilePhase/Diluent/Blank Acetonitrile: Water: 50:50 Volume of Injection 20.0 µl RID Flow Cell Temperature 55°C Column Oven Temperature 40°C Run Time 30 minutes Column HPLC ODS-3V Inertsil Column, (250 x 4.6 mm x 5µm) 2.4 Procedure 2.4.1 Standard Solution Preparation. Accurately weighed 100 mg of the cocamidopropyl betaine reference standard into a 25 mL volumetric flask. Added approximately 10 mL of diluent, then stopper the flask and vortex for about 30 minutes at 2500 RPM to ensure complete dissolution. Finally, made up the volume to 25 mL with the diluent. Filter the solution through a 0.45 µm PTFE syringe filter before transferring it to the HPLC vials. 2.4.2 Sample Solution Preparation. Weighed 2.5 g of the shampoo sample into a 50 mL Tarson tube. Added approximately 10 mL of diluent and vortex the mixture for about 30 minutes at 2500 RPM to dissolve the shampoo sample. Then, adjusted the volume to 25 mL with the diluent. Next, centrifuge sample for 10 minutes at 5000 RPM at 20°C. After centrifugation, carefully collect the clear upper layer using a dropper and filter it through a 0.45 µm PTFE syringe filter before transferring it to the HPLC vials. 2.4.3 Sample (Placebo) Preparation . Weighed 2.5 g of the sample (Without CAPB) into a 50 mL Tarson tube. Added approximately 10 mL of diluent and vortexed the mixture for about 30 minutes at 2500 RPM to fully dissolve the shampoo. The volume was then adjusted to 25 mL with additional diluent. Subsequently, the sample was centrifuged for 10 minutes at 5000 RPM at 20°C. After centrifugation, the clear upper layer was carefully collected using a dropper and filter it through a 0.45 µm PTFE syringe filter before being transferred to the HPLC vials. 3. Result and Discussion 3.1 Method Development and Optimization. In the first part of method development Mili-Q-water as diluent and mobile phase for analysing CAPB using HPLC with RI detector with C8 BDS column which encountered challenges like distorted peaks, uneven baseline, and interference from the placebo. To overcome these issues, modifications were made in mobile phase, and diluent. ACN is added to the mobile phase to achieve better separation, Additionally, a change in column was necessary to achieve better separation and well-resolved peaks for CAPB. After few trials with different solvents, two key changes were implemented: switching from water to diluent (Acetonitrile: water (50:50)) as the solvent and transitioning to an Inertsil ODS-3V C-18 column from previous C8 BDS column Vortex time and centrifuge time optimized for sample and standard preparation. Temperature plays a crucial role in RI-detector detection, 55ºC flow cell temperature has given better results from earlier 25ºC flow cell temperature. Under the above-optimized conditions, the retention time obtained for the CAPB is 7.3 minutes. (Fig. 3 ). Different shampoos, Shower gel and face wash were analysed using the developed method. CAPB contained in the samples were identified by comparing the retention times detected from samples with those of the standard CAPB (Table 3 ). Table 3 CAPB content in various Prototype Personal care Product Prototype %w/w CAPB % Recovery Shampoo 1. 2. 3. 2 97.2 2.5 96.9 1 98.0 Face Wash 1. 2. 3. 6 96.5 5.5 99.9 5 97.7 Shower Gel 1. 2. 3. 8 97.2 7 97.9 5 98.1 3.2 Analytical Method Validation . The method was developed following the guidelines set by the International Council for Harmonization (ICH) and AOAC International's Appendix K, which are crucial for ensuring the accuracy and reliability of analytical procedures. Validation Parameters performed as system suitability, System precision, Precision (repeatability), Recovery (Accuracy), Linearity, Solution Stability, Robustness, LOQ, LOD, and Ruggedness. All the above parameters were found within the limits. 3.2.1 System suitability : System suitability testing (SST) is an essential aspect of validating and controlling the quality of High-Performance Liquid Chromatography (HPLC) methods. It ensures that the entire analytical system including the instrument, column and sample preparation is operating effectively to deliver accurate and reliable results. By evaluating these parameters, the analyst can assess the performance of the HPLC system and to determine if it fulfils the required criteria for the analysis. System suitability is usually conducted at the start of the analysis or before each batch of samples. The tailing factor should not exceed 2, while the theoretical plate count was over 2,000. All these parameters were within acceptable limits (Table 4 ). Table 4 Standard Solution SST Name Retention time (min) Tailing factor Number of theoretical plates Cocamidopropyl betaine 7.28 1.117 6797 3.2.2 Specificity : Specificity is defined as the capability to precisely identify the analyte amidst other potential components, such as impurities and elements of the matrix. Specificity is performed by analysing sample containing analyte of analysis and various interferents or matrix components. To assess specificity, a comparison is conducted between the chromatograms obtained from the blank solution (Fig. 4 ), Standard CAPB (Fig. 3 ), placebo (Fig. 5 ) and sample containing the CAPB analytes of interest (Fig. 6 ). There should not be any interference at main analyte peak and at all known impurities from blank and placebo, there should not be any interference of known impurities with each other. For this we applied the same chromatographic conditions and injected it into HPLC-RI and the resulting chromatogram showed no co-eluting peaks at the eluting time of CAPB, indicating that the analyte peak was pure. 3.2.3 Linearity : The linearity of an analytical method refers to its ability to give test results that are directly proportional to the concentration of the analyte in samples within a specified range. Seven different concentrations (70%, 80%, 90%, 100%, 110%, 120% and 130%) of standard were analysed. Corresponding to concentration range of 2800 mg/L-5200 mg/L, respectively. L R 2 The correlation coefficient was calculated in duplicate, and its value comes out 0.9991 which is close to 1 that indicates high degree of linearity. In a linear regression line equation y = m * x + c, the slope is represented by the constant m, while the y-intercept is denoted by the constant c. (Figure_ 7) In summary, the provided information describes the linearity assessment of the analytical technique, where the linearity was evaluated by examining the relationship between peak area and analyte concentration using regression analysis and the linear regression line equation. 3.2.4 Precision : Precision is defined as the degree to which data values are close to one another across multiple measurements taken under the same analytical conditions. It encompasses three components: repeatability, intermediate precision, and reproducibility. The first type is system precision, or injection repeatability, which is assessed through multiple injections of a standard. This measurement reflects the HPLC instrument performance under the conditions mentioned in method. The second type of repeatability study is known as method precision, or analysis repeatability. In this each aliquot prepared independently according to the method procedure, within a single laboratory on the same day. The results for both system and method precision are within acceptable ranges. The standard deviation (SD) and relative standard deviation (RSD) were calculated for both solutions, results were falling within acceptable limits. An RSD of less than 2.0% for both system precision (Table 5 ) and method precision (Table 6 ) was deemed indicative of acceptable precision. Table 5 System precision data for the standard Injection No. Area STD CAPB 1 127533 2 127657 3 127735 4 127537 5 128247 6 127477 Average 127697.67 SD 285.1 %RSD 0.22 Table 6 Method precision data for the Sample solution Replicate No. CAPB (%w/w) Shampoo sample A 1.07 Shampoo sample B 1.10 Shampoo sample C 1.08 Shampoo sample D 1.12 Shampoo sample E 1.12 Shampoo sample F 1.09 Average 1.10 SD 0.02 %RSD 1.66 3.2.5 Recovery (accuracy) : Accuracy refers to the correctness of an analytical method, specifically the degree of agreement between the measured value and the actual value obtained. It is quantified as the percentage of analyte recovered from matrix components that have been spiked with a known quantity of components. Three different concentrations (80%, 100%, and 120%) of the standard solution were spiked, and analysis was carried out in triplicate. The percentage recovery is calculated using the formula [%Recovery= (Recovered concentration/Injected concentration) * 100]. For the method to be deemed acceptable, the average recovery should fall within the range of 90–110%. (Table 7 ) Table 7 Recovery data Spiked Sample % Recovered (CAPB) Shampoo sample A 80% 96.13 Shampoo sample B 80% 96.08 Shampoo sample C 80% 95.50 Average 95.9 %RSD 0.30 Shampoo sample A 100% 97.73 Shampoo sample B 100% 95.43 Shampoo sample C 100% 98.42 Average 97.2 %RSD 1.31 Shampoo sample A 120% 98.02 Shampoo sample B 120% 97.15 Shampoo sample C 120% 96.45 Average 97.2 %RSD 0.66 3.2.6 Robustness : Robustness is the ability of a method to remain same by small, deliberate changes in its parameters and this can be assessed by varying parameters like flow rate, pH of buffer/mobile phase, mobile phase composition, temperature, and wavelength etc., within an acceptable range. The quantitative impact of these variations is then evaluated. If the impact of the parameter remains unaffected from the specified limits, it is regarded as being within the method's robustness range. This was evaluated by changing the flow rate (mL/min), column oven temperature, refractive index detector temperature and in injection volume. No significant impact was noted. The relative standard deviation (RSD) for Cocoamidopropyl betaine was calculated from the peak area counts of duplicate injections under these varied conditions. The findings, summarized (Table 8 ), indicate that the method is robust against these minor chromatographic variations. Table 8 Robustness Data Request No Method precision value (CAPB) %w/w Observed value (CAPB) %w/w %RSD Change in flow rate 1.1ml 1.10 1.12 0.99 Change in flow rate 0.9ml 1.10 1.08 0.78 Change in Column oven temp 36ºC 1.10 1.09 0.50 Change in Column oven temp 44 ºC 1.10 1.09 0.50 Change in Refractive index detector temp 50 ºC 1.10 1.14 1.65 Change in Refractive index detector temp 60 ºC 1.10 1.13 1.39 Change in injection volume 22 µl 1.10 1.13 1.39 Change in injection volume 18 µl 1.10 1.10 0.0 3.2.7 Stability of sample solution : The stability of a sample solution is a crucial parameter, as it indicates the duration for which a solution can be stored before analysis without affecting the accuracy of the results. The stability of standard and sample was analysed by storing it at various temperatures. The sample solution was stored under the following conditions: 6 h at RT, 6 h at 2–8°C, 24 h at RT, 24 h at 2–8°C, 48 h at RT, and 48 h at 2–8°C. The assays of Cocoamidopropyl betaine were analysed (Table 9 ). Table 9 Stability of sample solution Stability Condition CAPB (%w/w) Shampoo Sample (Initial/RT) 1.12 Shampoo Sample (6Hr/RT) 1.10 Shampoo Sample (6Hr/2–8°C) 1.10 Shampoo Sample (24Hr/RT) 1.10 Shampoo Sample (24Hr/2–8°C) 1.10 AVERAGE 1.10 SD 0.01 %RSD 0.63 3.2.8 Ruggedness (Intermediate Precision) : Intermediate precision, also known as ruggedness, refers to the precision achieved when multiple analysts conduct the analysis using different instruments over several days within a single laboratory. Intermediate precision evaluates how consistent results are when a method is applied across various days, by different analysts, and using different instruments depending on available time and resources. Different analysts used Agilent 1260 Infinity II HPLC-RI systems to analyze the same sample. The method demonstrated ruggedness, as reproducible results were consistently obtained. (Table 10 ) Table 10 Intermediate precision data Replicate No. Cocamidopropyl betaine (%w/w) Shampoo sample A 1.07 Shampoo sample B 1.10 Shampoo sample C 1.08 Shampoo sample D 1.12 Shampoo sample E 1.12 Shampoo sample F 1.09 Average 1.10 SD 0.02 %RSD 1.66 3.2.9 LOD and LOQ : Limit of detection (LOD) represents the minimum concentration of analyte that can be detected, although it may not be quantifiable. The detection limit can be estimated by performing serial dilutions of an appropriate sample solution until the sample signal is no longer distinguishable from the blank signal. The limit of quantification (LOQ) is the minimum concentration of analyte that can be quantified with both accuracy and precision. It is established by decreasing the analyte concentration until it reaches a point where the method's precision becomes unacceptable. The limit of detection (LOD) and limit of quantification (LOQ) are two critical parameters in method validation. The LOD and LOQ were established based on a signal-to-noise ratio of 3:1 and 10:1, respectively, by diluting analyte solution with known concentrations. The LOD and LOQ values of Cocoamidopropyl betaine given below (Table 11 ). Table 11 LOD and LOQ Data Name LOD (µg/mL) S/N LOQ (µg/mL) S/N CAPB 60 3.51 200 12.2 4. Conclusion The study presents a novel, sensitive, and selective method for determining Cocamidopropyl Betaine (CAPB) in shampoo using reverse-phase high-performance Liquid Chromatography (RP-HPLC) coupled with a refractive index detector. The method validation results demonstrated that it was selective, precise, accurate, linear, and robust, with stability-indicating properties. The method has been successfully optimized to achieve excellent separation of CAPB. Furthermore, a key benefit of this method is that the mobile phase can be used as a diluent, leading to considerable time saving. Additional benefits include minimal sample preparation, short run times, and the availability of readily accessible detectors with low maintenance need. The method was validated under the ICH Q2 (R1) guidelines [ 23 , 24 ] and fulfilled all established validation parameters. For us, this method represented a considerable improvement over methods previously presented in papers on this subject. It is also eminently suitable for the use as a routine method in production quality control. Abbreviations CAPB, Cocamidopropyl betaine; HPLC, High Performance Liquid Chromatography; RI, Refractive index; LOD, Limit of detection; LOQ, Limit of quantification; RSD, Relative standard deviation; ICH, International Conference on Harmonization; AOAC, Association of Official Analytical Chemists; DMAPA, dimethylaminopropylamine; LAPDMA, lauramindopropyldimethlamine; NaCl, Sodium chloride; UV, ultraviolet; EBT, Eriochrome black T;RPM revolution per minute; Min, Minutes;R 2 , Correlation coefficient; SD, Standard deviation. Declarations Acknowledgement The author expresses gratitude to the Dabur Research and Development Centre, Dabur India Limited, for providing essential facilities and steadfast support throughout the study. Funding: There was no Funding. Consent to Publish declaration: We hereby consent to the publication of the manuscript titled “A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector ” in journal Discover Chemistry. We confirm that all the co-authors have agreed to the submission and publication. Ethics and Consent to Participate declarations: Not applicable Author Contribution declaration: Chetan and Yamini have performed all the experimental work, Chetan and Vishal draft the manuscript, Abdul provide the samples for analysis, Dr. S.K Luthra and Dr. Ranjan Mitra reviewed the manuscript. All the authors have read and approved the final manuscript. Competing Interest declaration: There are no Competing Interests. Data Availability Statement The data analyzed during the current study are available from the corresponding author on reasonable request. References P. Eichhorn, T.P. Knepper, J. Mass Spectrom. 36 (2001) 677–684. 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K. Luthra","email":"","orcid":"","institution":"Dabur India Limited","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"K.","lastName":"Luthra","suffix":""},{"id":498771714,"identity":"bb30567a-2757-42d6-b04c-897ed5f36844","order_by":4,"name":"Ranjan Mitra","email":"","orcid":"","institution":"Dabur India Limited","correspondingAuthor":false,"prefix":"","firstName":"Ranjan","middleName":"","lastName":"Mitra","suffix":""},{"id":498771715,"identity":"551147cc-6c17-4aa5-bc30-fc6dd391ec24","order_by":5,"name":"Vishal Sareen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACZgY2BoYCGK+CmcGAOC1wZWeI0cKArIWxjQgtBsfZnz34YcBgzz/78MHHhfOs5c0lch8w/KjYhlvLYR5zwx4DhsQZ59KSjWduSzfcOSPdgLHnzG2cWiSbedgkeAwYEhjO8JhJ8247zLjhRhoDM2MbPi3szyT/AB0mf4b/+2/eOYftCWrhZ2YwkwbawrjhDA8bM2/D4UQitADdI2MgkbjxDJuxNM+x9OSdPc8YDuLzCxv/8WeSbyps7OXOMD/8zFNjbbudPY3xwY8K3FqgQAKVe4CQ+lEwCkbBKBgF+AEAbZJKke7n1VcAAAAASUVORK5CYII=","orcid":"","institution":"Dabur India Limited","correspondingAuthor":true,"prefix":"","firstName":"Vishal","middleName":"","lastName":"Sareen","suffix":""}],"badges":[],"createdAt":"2025-07-13 14:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7113859/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7113859/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88917367,"identity":"8d011751-3d96-4da1-9de9-bfab949947a3","added_by":"auto","created_at":"2025-08-12 16:37:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":226255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/c91057784ce5a706bf4559b3.png"},{"id":88917364,"identity":"96c2503f-f0e1-4e75-9655-44e814270001","added_by":"auto","created_at":"2025-08-12 16:37:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44866,"visible":true,"origin":"","legend":"\u003cp\u003eThe synthetic procedures of CAPB\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/8a945e25e48385cfa49f6309.png"},{"id":88919719,"identity":"7c24d880-1b70-4b43-9ce1-edbec35a958c","added_by":"auto","created_at":"2025-08-12 17:01:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24995,"visible":true,"origin":"","legend":"\u003cp\u003eTypical chromatogram of CAPB standard\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/2bbb8ff2ef0b59dcf10723ec.png"},{"id":88918265,"identity":"f6477037-ae31-4a1d-9431-5c0fc07b9f7f","added_by":"auto","created_at":"2025-08-12 16:45:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23307,"visible":true,"origin":"","legend":"\u003cp\u003eTypical chromatogram of Blank\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/d2566ae996d2d00b09d0b47b.png"},{"id":88917365,"identity":"44d35a06-afee-4be0-b307-af4079e251b5","added_by":"auto","created_at":"2025-08-12 16:37:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26807,"visible":true,"origin":"","legend":"\u003cp\u003eTypical chromatogram of Placebo\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/d9fcebacb1d3ab1bf9d680fe.png"},{"id":88917371,"identity":"05907280-6a2f-436c-b463-e22cd3ba7396","added_by":"auto","created_at":"2025-08-12 16:37:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":27492,"visible":true,"origin":"","legend":"\u003cp\u003eTypical chromatogram of sample\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/6416ef055f12a4637bcac29b.png"},{"id":88917370,"identity":"a89ba35e-e413-44e8-80c6-2f80f600f54d","added_by":"auto","created_at":"2025-08-12 16:37:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24523,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve of Area vs. Concentration (mg/L) of the standard solution\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/32e2a16300b62fdd3d1ace93.png"},{"id":91326620,"identity":"ad791b83-ca2a-4486-a73f-0cdb06b83353","added_by":"auto","created_at":"2025-09-15 10:02:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1424718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7113859/v1/4450fb04-da80-4341-8fab-9e61796c90fc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector","fulltext":[{"header":"1. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Introduction\u003c/h2\u003e\u003cp\u003eSurfactants are a key group of compounds used in personal care products and detergents designed for both commercial and household applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. They have both hydrophilic and hydrophobic heads that reduce the surface tension between liquids-liquid/ liquid-solid/gas-liquid [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Surfactants are categorized on their head charge nature i.e. anionic, cationic, non-ionic and Zwitterionic surfactants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Isoelectric surfactants are molecules which have two ionic heads of opposite charge in single molecule [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Majority of the Zwitterionic surfactant are subtle to the human skin, which exhibits less lethal as within the same compound they have both negatively and positively charged structure and ensures cationic or anionic activity occurs within narrow pH range, avoiding both extremes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Various surfactants which have different heads are used simultaneously to improve product performance.\u003c/p\u003e\u003cp\u003eCocamidopropyl betaine (CAPB) is a moderate zwitterionic (amphoteric) ammonium-containing synthetic surfactant that is manufactured extensively and used in related industries products such as shampoos, contact lens solutions, detergents, skin care products, cleansers, liquid soap and antiseptics [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Johnson \u0026amp; Johnson was first company to introduce Cocamidopropyl betaine (CAPB) into personal care products in 1967 as \"No More Tears\" ingredient in children's shampoos. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As consumption continues to rise in personal care products like face wash, shampoo, etc. Natural products have struggled to keep up with their ongoing demand. Synthetic CAPB is one of the most widely used surfactant derivates derived from coconut oil commercially Cocamidopropyl betaine is frequently used in the manufacturing industry to cover the increasing demand for these products. The use of synthetic Cocamidopropyl betaine in personal care products can help to manage the rising demand and scarcity of natural resources. Coconut oil serves as a primary source of various types of fatty acids. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Coconut oil is mixture of fatty acids with diverse chain lengths and contains specific types of fatty acids, i.e.: lauric acid, myristic acid, caprylic acid and capric acid [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The fatty acids derived from coconut oil, either as a mixture or individually isolated, can be chemically modified with water loving substances to create surfactants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCAPB is produced by reacting fatty acids, frequently sourced from coconut oil or coconut with dimethylaminopropylamine (DMAPA), resulting lauramindopropyldimethlamine (LAPDMA) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. LAPDMA is consequently permitted to react with sodium monochloroaceate to give the product CAPB. CAPB has an anionic part (carboxylic group) and cationic part (quaternary ammonium group). The fat-loving group is formed by a combination of fatty acids of coconut with chain lengths between C-8 and C-18. This indicates that CAPB consists of compounds that share a common basic structure but differ in their lipophilic. Free fatty acids and sterols are contained by other substances, which are commercial products. CAPB is typically supplied as a moderately viscous solution, with concentrations typically ranging between 25%w/w to 30% w/w for personal care products. CAPB concentration is typically denoted by its activity level. It is mainly calculated as the total percentage of solids subtracted by the percentage of NaCl [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMixed surfactant system performs better than single surfactant system. Advance liquid detergents, personal care products are muti surfactant products with several interactions within the constituents that boost product effectiveness. Anionic and Amphoteric surfactants are basic surfactant used in personal care products to achieve outstanding detergency, cleansing quality, good foaming property and easy thickening in the presence of salt. CAPB has been the most important secondary surfactant for personal- cleansing products for a long time owing to the low irritation potential of the pure surfactant and its good toxicological properties with another anionic surfactant to reduces irritation to skin and mucus membrane and leads to a pleasant foam. CAPB is known to give a better cleansing performance and to improve the conditioning properties of hair. CAPB demand is increasing in personal care industries. Thus, accurately determining CAPB is crucial. In the past, various paper was published dealing with the solution of this problem using HPLC on different types of column and different detection techniques [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For surfactants, numerous analytical methods have been developed, including thin layer chromatography [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], titration with two-phase [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], HPLC on a cation exchange column [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Prep-HPLC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], Potentiometric analysis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], UHPLC\u0026ndash;MS/MS [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], ATR-FTIR[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These all-reported methods are highly dependent upon the matrix of the sample, or they required special type of column or sample pretreatment which require more time and large amount. If we talk about detectors like MS/MS, ATR_FTIR which are highly sophisticated detectors require high maintenance and high cost of analysis. Our method is substitute to these above methods. As far as we are aware, no method has been available based on HPLC coupled with a Refractive index on this column for the determination of CAPB in Shampoo. As RI detector effectively measures changes in the refractive index caused by the presence of CAPB in the sample, allowing accurate detection. RI detector maintenance is low, and cost of analysis is also cheap in comparison to MS and other detector. The column used is the simplest C18 column, which is readily available, cost effective, and does not require post- column treatment. The present study aims to develop and validate a new method based on HPLC-RI for exact determination of CAPB in Shampoo. The procedure underwent validation according to ICH guideline Q2 (R1).\u003c/p\u003e\u003c/div\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e\u003cp\u003eReference standard of Cocamidopropyl betaine (Lot no. 2-MJJ-54-1, Purity-35.7%w/w)) purchased from Toronto Research Chemical Canada, Acetonitrile purchased from Merck Limited (Lot no. DE4DF74840, Merck Life Science Pvt. Ltd. Mumbai, India) 0.45 \u003cem\u003e\u0026micro;\u003c/em\u003em PTFE syringe filter was procured from AXIVA (Lot no. SF0030119Axiva Sichem Pvt. Ltd. Sonipat, Haryana-131028, India). Water used was milli-Q-Water. Dabur vatika shampoo was taken for the analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Instrumentation and Equipment\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInstrument and equipment details\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInstrument/Equipment Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMake/Model\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnalytical Balance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXS105 dual range balance (Mettler Toledo)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMulti Tube Vortex Mixture (iSwix MV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNeuation Technologies Pvt. Ltd, 194/6/2, Dantali Industrial Estate, Gota -Vadsar Road Dantali, Kalol, Gandhinagar, Gujarat 382721Speed: 2500 RPM\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEppendorf Centrifuge\u003c/p\u003e\u003cp\u003e(5804 R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEppendorf India Private Limited, Plot No. 18,19, 20-(Part) South Phase, SIDCO Industrial Estate, Amb attur, Chennai, Tamil Nadu, India, Max Speed: 14000 rpm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHPLC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.Shimadzu, Model LC-2030C 3D, with refractive index detector model no. 20A, Made in Japan\u003c/p\u003e\u003cp\u003e2.Agilent Technologies, model 1200, with refractive index detector model no. 1260 infinity II, Made in Germany\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHPLC ODS-3V Inertsil Column, (250 x 4.6 mm x 5\u0026micro;m)\u003c/p\u003e\u003cp\u003eCatalogue. Number: 5020\u0026thinsp;\u0026minus;\u0026thinsp;01732, Serial Number: 20G0133298, made in Japan\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVolumetric flask\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25 mL class A(Borosil)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCentrifuge tube\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 mL (Tarson)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Chromatographic Condition\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHPLC Chromatographic Condition details\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDetector\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRefractive index detector (RID)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlow Rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0 ml/min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMobilePhase/Diluent/Blank\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAcetonitrile: Water: 50:50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVolume of Injection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.0 \u0026micro;l\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRID Flow Cell Temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn Oven Temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRun Time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30 minutes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHPLC ODS-3V Inertsil Column, (250 x 4.6 mm x 5\u0026micro;m)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Procedure\u003c/h2\u003e\u003cp\u003e\u003cb\u003e2.4.1 Standard Solution Preparation.\u003c/b\u003e Accurately weighed 100 mg of the cocamidopropyl betaine reference standard into a 25 mL volumetric flask. Added approximately 10 mL of diluent, then stopper the flask and vortex for about 30 minutes at 2500 RPM to ensure complete dissolution. Finally, made up the volume to 25 mL with the diluent. Filter the solution through a 0.45 \u0026micro;m PTFE syringe filter before transferring it to the HPLC vials.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.4.2 Sample Solution Preparation.\u003c/b\u003e Weighed 2.5 g of the shampoo sample into a 50 mL Tarson tube. Added approximately 10 mL of diluent and vortex the mixture for about 30 minutes at 2500 RPM to dissolve the shampoo sample. Then, adjusted the volume to 25 mL with the diluent. Next, centrifuge sample for 10 minutes at 5000 RPM at 20\u0026deg;C. After centrifugation, carefully collect the clear upper layer using a dropper and filter it through a 0.45 \u0026micro;m PTFE syringe filter before transferring it to the HPLC vials.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.4.3 Sample (Placebo) Preparation\u003c/b\u003e. Weighed 2.5 g of the sample (Without CAPB) into a 50 mL Tarson tube. Added approximately 10 mL of diluent and vortexed the mixture for about 30 minutes at 2500 RPM to fully dissolve the shampoo. The volume was then adjusted to 25 mL with additional diluent. Subsequently, the sample was centrifuged for 10 minutes at 5000 RPM at 20\u0026deg;C. After centrifugation, the clear upper layer was carefully collected using a dropper and filter it through a 0.45 \u0026micro;m PTFE syringe filter before being transferred to the HPLC vials.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result and Discussion","content":"\u003cp\u003e\u003cb\u003e3.1 Method Development and Optimization.\u003c/b\u003e In the first part of method development Mili-Q-water as diluent and mobile phase for analysing CAPB using HPLC with RI detector with C8 BDS column which encountered challenges like distorted peaks, uneven baseline, and interference from the placebo. To overcome these issues, modifications were made in mobile phase, and diluent. ACN is added to the mobile phase to achieve better separation, Additionally, a change in column was necessary to achieve better separation and well-resolved peaks for CAPB. After few trials with different solvents, two key changes were implemented: switching from water to diluent (Acetonitrile: water (50:50)) as the solvent and transitioning to an Inertsil ODS-3V C-18 column from previous C8 BDS column Vortex time and centrifuge time optimized for sample and standard preparation. Temperature plays a crucial role in RI-detector detection, 55\u0026ordm;C flow cell temperature has given better results from earlier 25\u0026ordm;C flow cell temperature. Under the above-optimized conditions, the retention time obtained for the CAPB is 7.3 minutes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDifferent shampoos, Shower gel and face wash were analysed using the developed method. CAPB contained in the samples were identified by comparing the retention times detected from samples with those of the standard CAPB (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCAPB content in various Prototype\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePersonal care Product\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrototype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e%w/w CAPB % Recovery\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003cp\u003e2.\u003c/p\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2 97.2\u003c/p\u003e\u003cp\u003e2.5 96.9\u003c/p\u003e\u003cp\u003e1 98.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFace Wash\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003cp\u003e2.\u003c/p\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6 96.5\u003c/p\u003e\u003cp\u003e5.5 99.9\u003c/p\u003e\u003cp\u003e5 97.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShower Gel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003cp\u003e2.\u003c/p\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8 97.2\u003c/p\u003e\u003cp\u003e7 97.9\u003c/p\u003e\u003cp\u003e5 98.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Analytical Method Validation\u003c/b\u003e. The method was developed following the guidelines set by the International Council for Harmonization (ICH) and AOAC International's Appendix K, which are crucial for ensuring the accuracy and reliability of analytical procedures. Validation Parameters performed as system suitability, System precision, Precision (repeatability), Recovery (Accuracy), Linearity, Solution Stability, Robustness, LOQ, LOD, and Ruggedness. All the above parameters were found within the limits.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.1 System suitability\u003c/b\u003e: System suitability testing (SST) is an essential aspect of validating and controlling the quality of High-Performance Liquid Chromatography (HPLC) methods. It ensures that the entire analytical system including the instrument, column and sample preparation is operating effectively to deliver accurate and reliable results. By evaluating these parameters, the analyst can assess the performance of the HPLC system and to determine if it fulfils the required criteria for the analysis. System suitability is usually conducted at the start of the analysis or before each batch of samples. The tailing factor should not exceed 2, while the theoretical plate count was over 2,000. All these parameters were within acceptable limits (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStandard Solution SST\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTailing factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNumber of theoretical plates\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCocamidopropyl betaine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6797\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.2 Specificity\u003c/b\u003e: Specificity is defined as the capability to precisely identify the analyte amidst other potential components, such as impurities and elements of the matrix. Specificity is performed by analysing sample containing analyte of analysis and various interferents or matrix components. To assess specificity, a comparison is conducted between the chromatograms obtained from the blank solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), Standard CAPB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), placebo (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and sample containing the CAPB analytes of interest (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere should not be any interference at main analyte peak and at all known impurities from blank and placebo, there should not be any interference of known impurities with each other. For this we applied the same chromatographic conditions and injected it into HPLC-RI and the resulting chromatogram showed no co-eluting peaks at the eluting time of CAPB, indicating that the analyte peak was pure.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.3 Linearity\u003c/b\u003e: The linearity of an analytical method refers to its ability to give test results that are directly proportional to the concentration of the analyte in samples within a specified range. Seven different concentrations (70%, 80%, 90%, 100%, 110%, 120% and 130%) of standard were analysed. Corresponding to concentration range of 2800 mg/L-5200 mg/L, respectively. L R\u003csup\u003e2\u003c/sup\u003e The correlation coefficient was calculated in duplicate, and its value comes out 0.9991 which is close to 1 that indicates high degree of linearity. In a linear regression line equation y\u0026thinsp;=\u0026thinsp;m\u003csub\u003e*\u003c/sub\u003ex\u0026thinsp;+\u0026thinsp;c, the slope is represented by the constant m, while the y-intercept is denoted by the constant c. (Figure_ 7)\u003c/p\u003e\u003cp\u003eIn summary, the provided information describes the linearity assessment of the analytical technique, where the linearity was evaluated by examining the relationship between peak area and analyte concentration using regression analysis and the linear regression line equation.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.4 Precision\u003c/b\u003e: Precision is defined as the degree to which data values are close to one another across multiple measurements taken under the same analytical conditions. It encompasses three components: repeatability, intermediate precision, and reproducibility. The first type is system precision, or injection repeatability, which is assessed through multiple injections of a standard. This measurement reflects the HPLC instrument performance under the conditions mentioned in method. The second type of repeatability study is known as method precision, or analysis repeatability. In this each aliquot prepared independently according to the method procedure, within a single laboratory on the same day. The results for both system and method precision are within acceptable ranges. The standard deviation (SD) and relative standard deviation (RSD) were calculated for both solutions, results were falling within acceptable limits. An RSD of less than 2.0% for both system precision (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and method precision (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) was deemed indicative of acceptable precision.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSystem precision data for the standard\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArea STD CAPB\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127533\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127657\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127735\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127537\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e128247\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127477\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127697.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e285.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMethod precision data for the Sample solution\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReplicate No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAPB (%w/w)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.5 Recovery (accuracy)\u003c/b\u003e: Accuracy refers to the correctness of an analytical method, specifically the degree of agreement between the measured value and the actual value obtained. It is quantified as the percentage of analyte recovered from matrix components that have been spiked with a known quantity of components. Three different concentrations (80%, 100%, and 120%) of the standard solution were spiked, and analysis was carried out in triplicate.\u003c/p\u003e\u003cp\u003eThe percentage recovery is calculated using the formula [%Recovery= (Recovered concentration/Injected concentration) * 100]. For the method to be deemed acceptable, the average recovery should fall within the range of 90\u0026ndash;110%. (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecovery data\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpiked Sample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% Recovered (CAPB)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample A 80%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e96.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample B 80%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e96.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample C 80%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample A 100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e97.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample B 100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample C 100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e98.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e97.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample A 120%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e98.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample B 120%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e97.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample C 120%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e96.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e97.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.6 Robustness\u003c/b\u003e: Robustness is the ability of a method to remain same by small, deliberate changes in its parameters and this can be assessed by varying parameters like flow rate, pH of buffer/mobile phase, mobile phase composition, temperature, and wavelength etc., within an acceptable range. The quantitative impact of these variations is then evaluated. If the impact of the parameter remains unaffected from the specified limits, it is regarded as being within the method's robustness range. This was evaluated by changing the flow rate (mL/min), column oven temperature, refractive index detector temperature and in injection volume. No significant impact was noted. The relative standard deviation (RSD) for Cocoamidopropyl betaine was calculated from the peak area counts of duplicate injections under these varied conditions. The findings, summarized (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), indicate that the method is robust against these minor chromatographic variations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRobustness Data\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRequest No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMethod precision value (CAPB)\u003c/p\u003e\u003cp\u003e%w/w\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eObserved value (CAPB)\u003c/p\u003e\u003cp\u003e%w/w\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in flow rate 1.1ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in flow rate 0.9ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in Column oven temp 36\u0026ordm;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in Column oven temp 44 \u0026ordm;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in Refractive index detector temp 50 \u0026ordm;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in Refractive index detector temp 60 \u0026ordm;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in injection volume 22 \u0026micro;l\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChange in injection volume 18 \u0026micro;l\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.7 Stability of sample solution\u003c/b\u003e: The stability of a sample solution is a crucial parameter, as it indicates the duration for which a solution can be stored before analysis without affecting the accuracy of the results.\u003c/p\u003e\u003cp\u003eThe stability of standard and sample was analysed by storing it at various temperatures. The sample solution was stored under the following conditions: 6 h at RT, 6 h at 2\u0026ndash;8\u0026deg;C, 24 h at RT, 24 h at 2\u0026ndash;8\u0026deg;C, 48 h at RT, and 48 h at 2\u0026ndash;8\u0026deg;C. The assays of Cocoamidopropyl betaine were analysed (Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStability of sample solution\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStability Condition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAPB (%w/w)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo Sample (Initial/RT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo Sample (6Hr/RT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo Sample (6Hr/2\u0026ndash;8\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo Sample (24Hr/RT)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo Sample (24Hr/2\u0026ndash;8\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAVERAGE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.8 Ruggedness (Intermediate Precision)\u003c/b\u003e: Intermediate precision, also known as ruggedness, refers to the precision achieved when multiple analysts conduct the analysis using different instruments over several days within a single laboratory. Intermediate precision evaluates how consistent results are when a method is applied across various days, by different analysts, and using different instruments depending on available time and resources.\u003c/p\u003e\u003cp\u003eDifferent analysts used Agilent 1260 Infinity II HPLC-RI systems to analyze the same sample. The method demonstrated ruggedness, as reproducible results were consistently obtained. (Table\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIntermediate precision data\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReplicate No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCocamidopropyl betaine (%w/w)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShampoo sample F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%RSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2.9 LOD and LOQ\u003c/b\u003e: Limit of detection (LOD) represents the minimum concentration of analyte that can be detected, although it may not be quantifiable. The detection limit can be estimated by performing serial dilutions of an appropriate sample solution until the sample signal is no longer distinguishable from the blank signal.\u003c/p\u003e\u003cp\u003eThe limit of quantification (LOQ) is the minimum concentration of analyte that can be quantified with both accuracy and precision. It is established by decreasing the analyte concentration until it reaches a point where the method's precision becomes unacceptable.\u003c/p\u003e\u003cp\u003eThe limit of detection (LOD) and limit of quantification (LOQ) are two critical parameters in method validation. The LOD and LOQ were established based on a signal-to-noise ratio of 3:1 and 10:1, respectively, by diluting analyte solution with known concentrations. The LOD and LOQ values of Cocoamidopropyl betaine given below (Table\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab11\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 11\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLOD and LOQ Data\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLOD (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS/N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLOQ (\u0026micro;g/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eS/N\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCAPB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe study presents a novel, sensitive, and selective method for determining Cocamidopropyl Betaine (CAPB) in shampoo using reverse-phase high-performance Liquid Chromatography (RP-HPLC) coupled with a refractive index detector. The method validation results demonstrated that it was selective, precise, accurate, linear, and robust, with stability-indicating properties. The method has been successfully optimized to achieve excellent separation of CAPB. Furthermore, a key benefit of this method is that the mobile phase can be used as a diluent, leading to considerable time saving. Additional benefits include minimal sample preparation, short run times, and the availability of readily accessible detectors with low maintenance need. The method was validated under the ICH Q2 (R1) guidelines [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and fulfilled all established validation parameters. For us, this method represented a considerable improvement over methods previously presented in papers on this subject. It is also eminently suitable for the use as a routine method in production quality control.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCAPB, Cocamidopropyl betaine; HPLC, High Performance Liquid Chromatography; RI, Refractive index; LOD, Limit of detection; LOQ, Limit of quantification; RSD, Relative standard deviation; ICH, International Conference on Harmonization; AOAC, Association of Official Analytical Chemists; DMAPA, dimethylaminopropylamine; LAPDMA, lauramindopropyldimethlamine; NaCl, Sodium chloride; UV, ultraviolet; EBT, Eriochrome black T;RPM revolution per minute; Min, Minutes;R\u003csup\u003e2\u003c/sup\u003e, Correlation coefficient; SD, Standard deviation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author expresses gratitude to the Dabur Research and Development Centre, Dabur India Limited, for providing essential facilities and steadfast support throughout the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThere was no Funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u0026nbsp;\u003c/strong\u003eWe hereby consent to the publication of the manuscript titled \u0026ldquo;A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector\u003cstrong\u003e\u0026rdquo;\u0026nbsp;\u003c/strong\u003ein journal Discover Chemistry. We confirm that all the co-authors have agreed to the submission and publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution declaration:\u003c/strong\u003e Chetan and Yamini have performed all the experimental work, Chetan and Vishal draft the manuscript, Abdul provide the samples for analysis, Dr. S.K Luthra and Dr. Ranjan Mitra reviewed the manuscript. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest declaration:\u0026nbsp;\u003c/strong\u003eThere are no Competing Interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed during the current study are available from the corresponding author on reasonable request. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP. Eichhorn, T.P. Knepper, J. Mass Spectrom. 36 (2001) 677\u0026ndash;684.\u003c/li\u003e\n\u003cli\u003eL.H. Levine, J.L. Garland, J.V. Johnson, J. Chromatogr. A 1062 (2005) 217\u0026ndash;225.\u003c/li\u003e\n\u003cli\u003eC.L. Yuan, Z.Z. Xu, M.X. Fan, H.Y. Liu, Y.H. Xie, T. Zhu, Study on characteristics and harm of surfactants, Journal of Chemical and Pharmaceutical Research, 6 (2014) 2233-2237.\u003c/li\u003e\n\u003cli\u003eD. Kwasniewska, D.Wieczorek, R. Zielinski, J. Surfactant Deterg. 18 (2015) 477\u0026ndash;486.\u003c/li\u003e\n\u003cli\u003eRoyal Society of Chemistry. Surfactants: the ubiquitous amphophiles. 2004. Available at: http://www.chemsoc.org/chembytes/ezine/2003/hargreaves_jul03.htm (accessed March 26, 2006).\u003c/li\u003e\n\u003cli\u003eColumbia Electronic Encyclopaedia. Detergent. 2000\u0026ndash;2005.Available at: http://www.infoplease.com/ce6/sci/A0815306.html (accessed Oct8, 2005).\u003c/li\u003e\n\u003cli\u003eSomasundaran P, Zhang L, Anjing L. Surfactants and interfacial phenomena in cosmetics and detergency. Cosmetics Toiletries 2001;116:53\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eJacob, Sharon E., and Sadegh Amini. \u0026quot;Cocamidopropyl betaine.\u0026quot; \u003cem\u003eDERM\u003c/em\u003e 19.3 (2008): 157-160.\u003c/li\u003e\n\u003cli\u003eBegoun P. Baby shampoo. In: Asmus S, Hopper J, Forbes Provo J, Parsons S, editors. Don\u0026rsquo;t go shopping for hair-care products without me. 3rd ed. Seattle, Washington: Beginning Press; 2004.p. 80.\u003c/li\u003e\n\u003cli\u003eZuo, Qi., Zhihui, Wang., Peng, Li., Luyuan, Yang. \u0026amp; Zhaozheng, Song. Studies on the Synthesis andApplication Properties of a Betaine Surfactant with a Benzene Ring Structure, Appl. Sci. 13, 4378(2023).\u003c/li\u003e\n\u003cli\u003eKatri, Suuronen., Maria, Pesonen. \u0026amp; Kristiina, Aalto-Korte. Occupational contact allergy tococamidopropyl betaineand its impurities. Contact Dermatitis. 66 (5), 286\u0026ndash;292 (2012). \u003c/li\u003e\n\u003cli\u003eChnuch, A., Lessmann, H., Geier, J. \u0026amp; Uter, W. Is cocamidopropyl betaine a contactallergen? Analysisof network dataand short review of the literature. Contact Dermatitis. 64, 203\u0026ndash;211 (2011).\u003c/li\u003e\n\u003cli\u003eC. Foti, D. Bonamonte, G. Mascolo, A. Corcelli, S. Lobasso, L. Rigano, G. Angelini,The role of 3-dimethylaminopropylamine and amidoamine in contact allergyto cocamidopropylbetaine, Contact Dermatitis 48 (4) (2003) 194\u0026ndash;198.\u003c/li\u003e\n\u003cli\u003eM.A. Liebert, J. Am. Coll. Toxicol. 10 (1991) 32\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eTegeler, Angela, Wolfgang Ruess, and Erich Gmahl. \u0026quot;Determination of amphoretic surfactants in cosmetic cleansing products by high-performance liquid chromatography on a cation-exchange column.\u0026quot; \u003cem\u003eJournal of Chromatography A\u003c/em\u003e 715.1 (1995): 195-198.\u003c/li\u003e\n\u003cli\u003eS.A. Bhawani, O. Sulaiman, R. Hashim, M.I.M.N. M., Analysis of Surfactants by Thin-Layer Chromatography A Review, 47 (2010) 73-80.\u003c/li\u003e\n\u003cli\u003eV.W. Reid, G.F. Longman, E. Heinerth, Electrochemical detection of surfactant cetylpyridinium bromide using boron-doped diamond as electrode, Tenside, 4(1967) 292-304.\u003c/li\u003e\n\u003cli\u003eTegeler, Angela, Wolfgang Ruess, and Erich Gmahl. \u0026quot;Determination of amphoretic surfactants in cosmetic cleansing products by high-performance liquid chromatography on a cation-exchange column.\u0026quot; \u003cem\u003eJournal of Chromatography A\u003c/em\u003e 715.1 (1995): 195-198.\u003c/li\u003e\n\u003cli\u003eNakamura, K., and Y. Morikawa. \u0026quot;Determination of surfactant mixtures in shampoos and detergents by HPLC.\u0026quot; \u003cem\u003eJournal of the American Oil Chemists Society\u003c/em\u003e 61.6 (1984): 1130-1135.\u003c/li\u003e\n\u003cli\u003eMadunić-Čačić, Dubravka, et al. \u0026quot;Potentiometric determination of anionic surfactants in formulations containing cocoamidopropyl betaine.\u0026quot; \u003cem\u003eInternational journal of electrochemical science\u003c/em\u003e 7.1 (2012): 875-885.\u003c/li\u003e\n\u003cli\u003eWang, Perry G., and Wanlong Zhou. \u0026quot;Rapid determination of cocamidopropyl betaine impurities in cosmetic products by core-shell hydrophilic interaction liquid chromatography-tandem mass spectrometry.\u0026quot; \u003cem\u003eJournal of Chromatography A\u003c/em\u003e 1461 (2016): 78-83.\u003c/li\u003e\n\u003cli\u003eCarolei, Luciano, and Ivano GR Gutz. \u0026quot;Simultaneous determination of three surfactants and water in shampoo and liquid soap by ATR-FTIR.\u0026quot; \u003cem\u003eTalanta\u003c/em\u003e 66.1 (2005): 118-124.\u003c/li\u003e\n\u003cli\u003eICH \u0026ldquo;Stability Testing of new Drug Substances and Products,\u0026rdquo; in Proceedings of the International Conference on Harmonization, \u003cstrong\u003eQ1a\u003c/strong\u003e, no. R2, 2003.\u003c/li\u003e\n\u003cli\u003eICH \u0026ldquo;Validation of analytical procedures: text and methodology,\u0026rdquo; in Proceedings of the International Conference on Harmonization, \u003cstrong\u003eQ2\u003c/strong\u003e, no. R1, 2005\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Shampoo, Cocamidopropyl betaine (CAPB), Validation, HPLC, RI","lastPublishedDoi":"10.21203/rs.3.rs-7113859/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7113859/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCocamidopropyl betaine (CAPB) is a moderate zwitterionic (amphoteric) synthetic surfactants commonly used in shampoos and other personal care products. They serve as key ingredients that help to cleanse, emulsify, and create foam, removing dirt, impurities from the skin and hair. CAPB has been increasingly used in personal care formulations because of its foam boosting and high mildness properties. This study aimed to develop and validate method for the quantification of CAPB in shampoo using HPLC-RI detector. All validation parameters were conducted in compliance with the ICH guidelines and AOAC International Appendix K and were found to be within permissible limits. R\u003csup\u003e2\u003c/sup\u003e for linearity was greater than 0.99. LOD and LOQ were 60 µg/mL and 200 µg/mL respectively. The % RSD for method precision was 0.85%. The recovery range was found within 95–98%. The developed method is precise, robust, independent of the Shampoo matrix and thus suitable for routine for CAPB identification and quantification in Shampoo.\u003c/p\u003e","manuscriptTitle":"A novel method for the quantification of Cocamidopropyl betaine through High Performance Liquid Chromatography coupled with a Refractive Index Detector","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 16:37:41","doi":"10.21203/rs.3.rs-7113859/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":"40cbcfe8-edb3-4514-bdba-f071a571dd4f","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T09:53:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-12 16:37:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7113859","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7113859","identity":"rs-7113859","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}