Concurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets

Concurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets | 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 Concurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets Krishnaphanisri Ponnekanti, Ramreddy Godela This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4892587/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract The present research aims to establish new stability indicating RP-UPLC method for concurrently estimating Pregabalin and Etoricoxib in blended powder and their combined tablet formulation. The effective separation of Pregabalin and Etoricoxib achieved with HSS column C18 (150x2.1mm,1.8µm), 0.1% orthophosphoric acid: acetonitrile in 65:35 v/v) at a flow rate of 0.3mL/min, and isocratic elution at 228nm. The elution of PRB and ETB was noticed at 1.56 and 2.01 minutes, with good resolution and system suitability with the developed approach. PRB and ETB have shown linear responses from 18.75 to 112.5µg/mL and 15 to 90µg/mL, respectively. The range of the % RSD for intraday and inter-day precision was 0.33 to 0.81. The LOD and LOQ of Pregabalin and Etoricoxib were computed to be 0.07 µg/mL, 0.21 µg/mL, and 0.01µg/mL, 0.04 µg/mL, respectively, by standard deviation method. The validation method was carried out using ICH standards. The stability- indicating the feature of the method was confirmed by the forced degradation studies where degradants generated by stress testing were clearly distinguished from the peaks of analytes. The shorter elution period and superior sensitivity of both analytes with this method are appropriate for regular analysis of Pregabalin and Etoricoxib. Pregabalin Etoricoxib RP-UPLC isocratic elution Stability indicating C18 column Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Pregabalin (PGB) is an anticonvulsant and analgesic agent [ 1 ]. It is well tolerated for managing neuropathic pain and chronic pain conditions [ 2 ]. It has a wide safety margin. PGB is a first-line or adjunctive therapy for many conditions, including generalized anxiety disorders, diabetic peripheral neuropathy, and partial seizures [ 3 – 6 ]. Etoricoxib (ETB) is an NSAID and COX-2 inhibitor [ 7 ]. This is primarily used to treat people with gout, osteoarthritis, rheumatoid arthritis, and spondylitis-related joint pain and swelling [ 8 – 10 ]. It is also employed to lessen edema and stiffness in the joints [ 11 ]. A Fixed-Dose Combination (FDC) is a product that combines two or more active ingredients in a fixed ratio and is used for treatment by integrating drugs, devices, or biological products. Defined by the U.S. FDA, FDCs enhance compliance, effectiveness, and cost-effectiveness while reducing pill burden and side effects [ 12 ]. They are widely prescribed globally for managing complex diseases such as HIV/AIDS, diabetes, hypertension, tuberculosis, neuropathic pain, asthma, and malaria. This approach streamlines treatment regimens, making them more efficient and patient-friendly. The chemistry of the PRB and ETB is illustrated in Fig. 1 [ 13 , 14 ]. The combination of PRB and ETB provides good therapeutic benefits compared to a single regimen treatment in pinched nerves with muscle weakness [ 15 ]. Analytical methods are essential for accurately measuring drug amounts and purity, ensuring they are safe and effective. They check the quality of medicines, find impurities, and meet regulatory standards. These methods help develop new drugs by providing essential data. They also monitor drug levels in patients for proper dosing and fewer side effects. Overall, they ensure that medicines work correctly and safely. As of now, numerous UV techniques, chromatographic methods, and hyphenated methods like LC-MS and LC-MS/MS have been reported for PGB as a single drug moiety or in combination with other drugs [ 16 – 23 ]. Similarly, ETB possesses different analytical methods for identification and estimation [ 24 – 30 ]. Few HPLC methods were reported in the literature for simultaneous estimation of PRB and ETB [ 31 – 34 ]. In the reported methods, only some possess longer ETB retention time [ 31 , 32 ]. Few methods need stability-indicating properties [ 33 , 34 ]. UPLC method is superior to HPLC in terms of lower RT, improved peak resolution, efficiency of peaks, and less solvent consumption due to less particle size of stationary phase and high-pressure pumps [ 35 ]. 2. MATERIALS AND METHODS PRB and ETB in pure form were purchased from Spectrum Pharma in Hyderabad. All analytical and HPLC grade solvents were purchased from a nearby Merck India Limited vendor. Waters UPLC (TUV detector and Empower-2 software) was used to develop the method. Water (Milli-Q) and a 1 mg sensitive balance (SCALETEC-SAB224CL) were also utilized. 2.1 Method development The method development was done using a trial-and-error concept. Different polar solvents with different rations were used to optimize the method by considering system suitability as per ICH. The effective separation of PRB and ETB was achieved with HSS column C18 (150x2.1mm,1.8µm), 0.1% orthophosphoric acid: acetonitrile in 65:35 v/v) at a flow rate of 0.3mL/min, isocratic elution at 228nm, and a column temperature of 30 0 C. Equal volumes of acetonitrile and water were used as a diluent to prepare the working standard, sample, and forced degradation solution. 2.1.1 Preparation of standard solution Accurately transferred about 75mg of PRB and 60mg of ETB pure powders into a volumetric flask measuring 100 mL to obtain750 µg/mL and 600 µg/mL for PRB and ETB. 1mL of the resultant solution was further made to 10 mL with diluent in a 10 mL calibrated volumetric flask to produce concentrations of 75 µg/mL and 60 µg/mL of PRB and ETB, respectively, which is referred to as 100% level concentration. 2.1.2 Preparation of sample solution The tablet powder equivalent 75 mg of PRB and 60 mg of ETB pure powders into a volumetric flask measuring 100 mL to obtain750 µg/mL and 600 µg/mL for PRB and ETB. 1mL of the resultant solution was further made to 10 mL with diluent in a 10 mL calibrated volumetric flask to produce concentrations of 75µg/mL and 60 µg/mL of PRB and ETB, respectively. The sample solution was filtered through the 0.45µ filter to exclude the particulate matter. Method development 2.2 Method validation The validation of the method has been performed with the provision of ICHQ2R1 guidelines [ 35 – 37 ]. 2.2.1 System suitability Six replicate injections of a standard solution consisting of PRB (75µg/ mL) and ETB (60 µg/mL) were analyzed by the UPLC system with optimized conditions. The ICH recommended system suitability parameters, including theoretical plates, tailing factor, %RSD, and resolution, were determined to ensure the method's suitability toward the UPLC system. 2.2.2 Linearity The Linearity of the proposed method was established for PRB and ETB in the range of 18.75µg/mL to 112.5µg and 15µg/mL to 90µg/mL, respectively. A linear curve between concentration and peak area is used to get linear regression data to assess the slope, intercept, and regression coefficient(R 2 ). The concentrations of about 18.75, 37.5,56.25,75,93.75 and 112.5µg/mL of PRB and 15,30,45,60,75 and 90µg/mL of ETB were analyzed in triplicate by the proposed approach to ensure the linearity range. 2.2.3 Accuracy The proposed UPLC method’s accuracy was ascertained by the standard addition method, where different levels (50%, 100%, and 150%) of standard solutions were separately spiked to a known amount of sample solution. The average (n = 3) percentage recovery of various levels of standard solution from different spiked solutions was calculated from the recorded chromatograms. 2.2.4 Precision Precision is referred to as the reliability of the observed responses of a homogeneous sample over numerous replications. In most cases, Precision should be done on the same day (intraday) and distinct days(inter-day) as per ICH recommendations. Six repetitive injections of standard solution in a day and three repetitions per day for three consecutive days were analyzed to perform both intraday and inter-day precision. The %RSD was calculated for the peak areas that were obtained. 2.2.5 Robustness The ability of the approach to withstand altering the parameters of the method in the analysis of the intended analyte without any changes in the analytical results. In the present approach, deliberate changes have been made to the mobile phase (± 1mL), column temperature (± 5 o C), and flow rate (± 0.1mL/min). The system suitability parameters of chromatograms were obtained for standard solutions by injecting each altered parameter in triplicate. 2.2.6 Specificity The method's capacity to recognize the selected drug in the presence of other substances is referred to as specificity. Individual injections of 1µL of prepared blank solution, working standard solution, sample solution, and forced degradation solution were injected to detect any interference with the peaks of PRB and ETB from other peaks in the acquired chromatograms. 2.2.7 Sensitivity Sensitivity in terms of Limit of Detection (LOD) and Limit of quantification (LOQ) was assessed by the standard deviation (SD) method, where the SD of the intercept and average slope of the linear curve (n = 3) were considered. LOD = 3.3 σ/S LOQ = 10 σ/S σ- SD of intercept of the linear plot, S- the average slope of the linear plot (n = 3) 2.3 Forced degradation (FD) studies FD studies HPLC are intended to develop the stability-indicating method where stability and degradation of the intended analyte can be determined [ 38 ]. These FD studies help to assess the degradation behaviour and storage conditions to ensure drug safety and efficacy as per ICH requirements. These studies used the ICH Q1A(R2) and Q1B provisions [ 39 – 41 ]. The stability of PRB and ETB was examined under various FD conditions. In acid hydrolysis, 1mL of 2N HCl was mixed with 1mL of the standard stock solution, refluxed for 30 minutes at 60°C, and neutralized with the same strength NaOH. The resultant solution was diluted in such a way as to attain 75µg/mL and 60µg/mL of PRB and ETB, respectively. Base hydrolysis shadowed a similar process with 2N NaOH. Similarly, 20% H 2 O 2 was used in oxidative degradation. All three samples were kept aside for 48 hr. while thermal degradation involved exposing the stock solution for 72 hr at 105°C/75% RH. Photostability was determined by exposing the standard stock solution to UV light in the dark control for 7 days. In neutral degradation, equal portions of HPLC-grade water and standard stock solution were mixed uniformly. In each case, further dilution was done to get a solution of 75µg/mL of PRB and 60µg/mL of ETB. Each solution was analyzed in triplicate to determine the percentage degradation of PRB and ETB. 2.4 Assay The % purity of the commercial formulation can be assessed using an assay. It was carried out by injecting standard and sample solutions containing 75µg/mL of PRB and 60µg/mL of ETB, respectively. 3. RESULTS AND DISCUSSION 3.1 Optimized method The method development starts with initial conditions such as HSS column C18 (150x2.1mm,1.8µm), methanol: water in 50:50 v/v) at a flow rate of 0.3mL/min, isocratic elution at 228nm, and a column temperature of 30 0 C. A broad peak with a low plate count was observed for ETB. A further modification was made by replacing water in the state mobile phase with KH2PO4, which produced broad peaks for both PRB and ETB. Finally, a mobile phase of 0.1% OPA and acetonitrile (65:35 v/v) provided sharp and efficient peaks with plate counts of more than 2000. The retention time of PRB and ETB was noticed at 1.56 and 2.01 minutes, with good resolution and system suitability with the developed approach (Figure-2). 3.2 Method validation 3.2.1 System suitability The six replicate injections of the standard solution (PRB − 75µg/mL and ETB − 60 µg/mL) resulted in an average USP plate count more fabulous than 2000 (i.e., 2980) and a tailing factor of 2 or less (i.e., 1.4). The % RSD of peak response for PRB and ETB is two or less(i.e.,0.30). The mean resolution between PRB and ETB in the chromatographic peaks is 6.2, more significant than two. All the system suitability parameters results obeyed the ICH recommended limits (Table 1 ). Table 1 Results of system suitability of the developed RP-UPLC method. Drug name Statistical parameter RT (Min) Peak response USP Plates Tailing factor Resolution PRB Mean (n = 6) 1.56 818272.67 2353.00 1.41 - SD 0.01 6765.03 109.95 0.04 - %RSD 0.50 0.83 4.67 2.61 - ETB Mean (n = 6) 2.04 785714.33 2814.67 1.24 6.2 SD 0.02 2403.16 95.54 0.01 0.13 %RSD 0.11 0.03 0.34 0.06 0.21 3.2.2 Linearity The developed method has shown significant linearity with an R² value of 0.999 for the concentration ranges of PRB (18.75µg/mL to 112.5µg/mL) and ETB (15µg/mL to 90µg/mL)). These results reveal that the established method's linearity aligns with ICH recommendations. The obtained chromatogram showing this linearity is shown in Table 2 and Fig. 3. Table 2 Results of linearity of PRB and ETB by developed method. S. No PRB ETB Concentration (µg/mL) Peak area Concentration (µg/mL) Peak area 1 18.75 203260 15 201015 2 37.5 400240 30 416912 3 56.25 610155 45 622526 4 75 820299 60 826289 5 93.75 1020461 75 1031996 6 112.5 1230021 90 1235425 (Correlation coefficient) R² 0.999 0.999 3.3.3 Accuracy The average percentage recovery of PRB and ETB in 50%, 100%, and 150% levels of spiked solutions was calculated to be in the range of 99.0–99.845% (Table 3 ). These results were within 100% ± 2 limits as per ICH recommendations. Table 3 Results of percentage recovery of PRB and ETB. Drug name % Level Amount of standard solution *% Recovery Added (µg/mL) Recovered (µg/mL) PRB 50 37.5 37.2 99.2 100 75 74.3 99.0 150 112.5 112.1 99.64 ETB 50 30 29.72 99.0 100 60 59.61 99.35 150 90 89.87 99.85 *Mean of three determinations 3.3.4 Precision Intraday and inter-day precision found that the computed % RSD values of the peak area responses standard solution of PRB and ETB in both intraday and inter-day precision were observed to be in the range of 0.27 to 0.55 (Table-4). The %RSD value less than or equal to two emphasizes the considerable precision of the established method. Table 4 Intraday and inter-day results of PRB and ETB in standard solution. Type of precision Peak area Parameter PRB 75 (µg/mL) ETB 60 (µg/mL) Intraday Mean ± SD (n = 6) 826041.5 ± 2274.95 784085.7 ± 2274.95 %RSD 0.27 0.42 Inter-day Mean ± SD (n = 9) 817685.8 ± 4549.40 785597.8 ± 3624.8 %RSD 0.55 0.46 3.3.5 Robustness The slight and intentional alterations in the mobile phase, flow rate, and column temperature could not affect the method's performance. The results of the obtained system suitability parameters complied with ICH guidelines (Table 5 ), confirming the developed method's robustness. Table 5 System suitability parameter data of PRB and ETB in the method's robustness. Variation of Parameter Pregabalin(n = 6) Peak Area (Mean ± SD) %RSD Plate count Tailing Factor Mobile phase ratio (± 1mL) 66:34 820830 ± 6509.8 0.8 2599 1.21 64:36 815294 ± 4901.2 0.6 2562 1.28 Flow rate (± 0.1mL) 0.2 816438 ± 3517.0 0.4 2443 1.35 0.4 816820 ± 3868.9 0.5 2578 1.29 Temperature (± 5ºC) 25 818876 ± 5841.5 0.7 2582 1.31 35 816538 ± 6349.9 0.8 2544 1.28 Etoricoxib (n = 6) Mobile phase ratio (± 1mL) 66:34 819353 ± 4884.5 0.6 2855 1.19 64:36 820997 ± 5994.3 0.7 2808 1.19 Flow rate (± 0.1mL) 0.2 820276 ± 5772.2 0.7 2964 1.21 0.4 823831 ± 4652.9 0.6 2893 1.21 Temperature (± 5ºC) 25 821884 ± 8372.1 1.0 2806 1.20 35 813344 ± 2297.9 0.3 2829 1.20 3.3.6 Sensitivity The LOD and LOQ of PRB and ETB were computed to be 0.07 µg/mL, 0.21 µg/mL, and 0.01µg/mL, 0.04 µg/mL, respectively, by standard deviation method. 3.3.7 Specificity No interference from the placebo, blank, and degradant solution was found at the Pregabalin and Etoricoxib retention times (Fig. 4). It shows that the method is specific for estimating PRB and ETB under the stated method conditions. 3.4 Forced degradation studies The percentage degradation of PRB and ETB in degradation solutions was determined by comparing the peak responses of both analytes in freshly prepared standard solution with that of forced degradation solutions. The percentage degradation of PRB is higher in acidic conditions and very low in neutral degradation studies. Similarly, ETB has shown higher degradation in photolytic conditions and very low degradation in neural conditions. The above results significantly confirm the susceptibility of PRB and ETB toward acidic and photolytic environments, respectively (Fig. 5). The degradation of PRB and ETB in stated degradation conditions was identified by the developed approach regarding reduced peak response, and degradant peaks ensure the stability indicating feature of the established RP-UPLC method. 3.5 Assay The percentage purity of PRB and ETB in the marketed tablet was determined to be 99.92 ± 0.52% and 99.86 ± 0.61%, respectively (Table 6 ). Table 6 Percentage purity of PRB and ETB in commercial tablet. Drug name RT (Min) Areas Tailing factor Plate count Assay ± SD (%w/w) (n = 6) PRB Standard 1.55 817249 1.42 2365 99.92 ± 0.52 sample 1.54 818637 1.40 2355 ETB Standard 2.01 787484 1.23 2806 99.86 ± 0.61 sample 2.00 789479 1.20 2877 The developed UPLC method exerts advantages over the reported methods. No single UPLC method is available in the literature for determining PRB and ETB in combined dosage forms. In the reported HPLC methods, longer RT is needed, and ETB is needed more than 5 minutes [ 31 , 32 ]. The RT of PRB and ETB in the current method is 1.5 and 2.0 min in that order. The LOD and LOQ values of the current approach were highly superior to the existing methods [ 31 – 34 ]. Stability-indicating features for HPLC or UPLC is highly significant in current scenarios in analytical methods to adopt the methods in regular analysis. All the reported methods lacked stability, indicating property, compared to the current method [ 32 – 34 ]. The stated validation forced degradation studies results of the current method were aligned with the recommendations of ICH guidelines. The above results emphasize the developed approach's efficacy, compatibility, and economic aspects. 4. CONCLUSION An economical, robust, sensitive, and specific reverse-phase liquid chromatography approach was established, offering improved sensitivity and sample analysis time. The developed method was validated and verified under varied stress conditions, ensuring stability-indicating property and specificity towards PRB and ETB. This approach magnificently distinguishes PRB and ETB, along with their potential degradants, with high resolution. The shorter elution period and superior sensitivity of both analytes with this method are appropriate for regular analysis of PRB and ETB, confirming reliable results and effective separation. FUTURE PROSPECTIVE RESEARCH In the current research study, the UPLC stability-indicating method was established. This method is highly effective in routinely analyzing the mentioned drugs in manufacturing units' bulk and dosage forms. To know the nature of degradants and impurities, further investigation of pharmaceuticals should be conducted using advanced hyphenated techniques like LC-NMR and LC-MS/MS. Abbreviations ETB- Pregabalin PRB- Etoricoxib FD- Forced Degradation ICH- International Committee for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use RP- HPLC- Reverse Phase High-Performance Liquid Chromatography LOD: Limit of Detection LOQ: Limit of Quantification RSD: Relative Standard Deviation SD: Standard Deviation RT: Retention Time Declarations AUTHORS’ CONTRIBUTIONS All the authors contributed equally to the design and frame of the work, acquisition, interpretation of data, and manuscript preparation; all authors have read the prepared manuscript and approved it for publication. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Not applicable. HUMAN AND ANIMAL RIGHTS Not applicable. CONSENT FOR PUBLICATION Not applicable. AVAILABILITY OF DATA AND MATERIALS The data and supportive information are available within the article. FUNDING None. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS The authors are thankful to the GITAM School of Pharmacy, GITAM Deemed to be University, and the Malla Reddy Institute of Pharmaceutical Sciences. References Toth C. Pregabalin: latest safety evidence and clinical implications for the management of neuropathic pain. Therapeutic advances in drug safety. 2014 Feb;5(1):38-56. Ben‐Menachem E. Pregabalin pharmacology and its relevance to clinical practice. Epilepsia. 2004 Aug;45:13-8. Kavoussi R. Pregabalin: from molecule to medicine. European Neuropsychopharmacology. 2006 Jul 1;16:S128-33. Dworkin RH, Kirkpatrick P. Pregabalin. Nature reviews Drug discovery. 2005 Jun 1;4(6):455-7. Lauria-Horner BA, Pohl RB. Pregabalin: a new anxiolytic. Expert opinion on investigational drugs. 2003 Apr 1;12(4):663-72. Shneker BF, McAuley JW. Pregabalin: a new neuromodulator with broad therapeutic indications. Annals of Pharmacotherapy. 2005 Dec;39(12):2029-37. Cochrane DJ, Jarvis B, Keating GM. Etoricoxib. Drugs. 2002 Dec;62:2637-51. Takemoto JK, Reynolds JK, Remsberg CM, Vega-Villa KR, Davies NM. Clinical pharmacokinetic and pharmacodynamic profile of etoricoxib. Clinical pharmacokinetics. 2008 Nov;47:703-20. Dallob A, Hawkey CJ, Greenberg H, Wight N, De Schepper P, Waldman S, Wong P, DeTora L, Gertz B, Agrawal N, Wagner J. Characterization of etoricoxib, a novel, selective COX‐2 inhibitor. The Journal of Clinical Pharmacology. 2003 Jun;43(6):573-85. Martina SD, Vesta KS, Ripley TL. Etoricoxib: a highly selective COX-2 inhibitor. Annals of Pharmacotherapy. 2005 May;39(5):854-62. Patrignani P, Capone ML, Tacconelli S. Clinical pharmacology of etoricoxib: a novel selectiveCOX2 inhibitor. Expert Opinion on Pharmacotherapy. 2003 Feb 1;4(2):265-84. Kim DW, Weon KY. Pharmaceutical application and development of fixed-dose combination: Dosage form review. Journal of Pharmaceutical Investigation. 2021 Sep;51:555-70. PubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 5486971, Pregabalin; [cited 2024 July 30]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Pregabalin PubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 5486971, Pregabalin; [cited 2024 July 30]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Pregabalin Yeole AB, Sree Ranga Lakshmi G, Selvakumar CJ, Goni VG, Nawal CL, Valya BJ et al., Efficacy and Safety of Pregabalin Prolonged Release-Etoricoxib Combination Compared to Etoricoxib for Chronic Low Back Pain: Phase 3, Randomized Study. Pain Ther. 2022 Dec;11(4):1451-1469. Kasawar GB, Farooqui MN. Development and validation of HPLC method for the determination of pregabalin in capsules. Indian journal of pharmaceutical sciences. 2010 Jul;72(4):517. Pingale P, Singasane T. Development and validation of HPLC method for the determination of pregabalin in bulk and in pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2012;5(6):829-33. Martinc B, Roškar R, Grabnar I, Vovk T. Simultaneous determination of gabapentin, pregabalin, vigabatrin, and topiramate in plasma by HPLC with fluorescence detection. Journal of Chromatography B. 2014 Jul 1;962:82-8. Arayne MS, Shahnaz H, Ali A, Sultana N. Monitoring of Pregabalin in pharmaceutical formulations and human serum using UV and RP-HPLC techniques: Application to dissolution test method. Pharm Anal Acta. 2014;5(287):2. Radwan RA, Abouzied NF, Badway FA, HA H, Hassan AM. New Method Development for Pregabalin Detection in Human Plasma by HPLCDAD. J. Pharm. Appl. Chem. 2020;6(3):61-. Bhatt KK, Patelia EM, Mori A. Simultaneous estimation of pregabalin and methylcobalamine in pharmaceutical formulation by RP-HPLC method. Journal of Analytical and Bioanalytical Techniques. 2013;4(1):1-4. Kannapan N, Nayak SP, Venkatachalam T, Prabhakaran V. Analytical RP-HPLC method for development and validation of pregabalin and methylcobalamine in combined capsule formulation. Kavitha MP, Rajasekhar A. A validated HPLC method for the analysis of pregabalin and methylcobalamin in bulk and pharmaceutical formulation. Pharmacie Globale. 2013 Jul 1;4(7):1. Patel HM, Suhagia BN, Shah SA, Rathod IS. Determination of Etoricoxib in Pharmaceutical Formulations by HPLC Method. Indian Journal of Pharmaceutical Sciences. 2007 Sep 1;69(5). Topalli S, Chandrashekhar TG, Annapurna MM. Validated RP‐HPLC Method for the Assay of Etoricoxib (A Non‐Steroidal Anti‐Inflammatory Drug) in Pharmaceutical Dosage Forms. Journal of Chemistry. 2012;9(2):832-8. Hartman R, Abrahim A, Clausen A, Mao B, Crocker LS, Ge Z. Development and validation of an HPLC method for the impurity and quantitative analysis of etoricoxib. Journal of liquid chromatography & related technologies. 2003 Aug 1;26(15):2551-66. Pattan SR, Jamdar SG, Godge RK, Dighe NS, Daithankar AV, Nirmal SA, Pai MG. RP-HPLC method for simultaneous estimation of paracetamol and etoricoxib from bulk and tablets. Journal of Chemical and Pharmaceutical Research. 2009 Jan;1(1):329-35. Kumar S, Joshi A, Thakur RS, Pathak AK, Shah K. Simultaneous estimation of etoricoxib and thiocolchicoside by RP-HPLC method in combined dosage forms. Acta Poloniae Pharmaceutica. 2011 Nov 1;68(6):839-43. Mandal U, Senthil Rajan D, Bose A, Gowda KV, Ghosh A, Pal TK. Development and Validation of an HPLC Method for Analysis of Etoricoxib in Human Plasma. Indian journal of pharmaceutical sciences. 2006 Jul 1;68(4). Alzweiri M, Sallam M, Al-Zyoud W, Aiedeh K. Stability study of etoricoxib a selective cyclooxygenase-2 inhibitor by a new single and rapid reversed phase HPLC method. Symmetry. 2018 Jul 17;10(7):288. Karanjkar MB. Development And Validation Of RP-HPLC For Simultaneous Estimation of Pregabalin and Etoricoxib in Pharmaceutical Tablet Dosage Form. IJPSR. 2021;13(4):872-9. Yeluri RR, Reddy BS, Kumari RR. Quantification of Pregabalin and Etoricoxib Combo in Tablets and Bulk with Developed RP-HPLC Method: Stability Indicating Feature Assessment. Journal of Advanced Scientific Research. 2022 May 30;13(04):31-6. Hanumanth V, Kumar PR, Sravya K, Padmavathi Y, Babu NR, Sowmya K. Simultaneous Estimation of Pregabalin and Etoricoxib in Bulk and Pharmaceutical Dosage Form by Using RP-HPLC Method. Journal of Drug Delivery and Therapeutics. 2023 Sep 15;13(9):20-7. Chaudhary A, Singh BK. Simultaneous Estimation of Pregabalin and Etoricoxib using Novel HPLC Method: An Application in Quantitative Analysis of Pharmaceutical Dosage Forms. Indian Journal of Pharmaceutical Education and Research. 2021 Jul 1;55:191. Nováková L, Matysová L, Solich P. Advantages of application of UPLC in pharmaceutical analysis. Talanta. 2006 Jan 15;68(3):908-18. Ravisankar P, Navya CN, Pravallika D, Sri DN. A review on step-by-step analytical method validation. IOSR J Pharm. 2015 Oct;5(10):7-19. Sharma S, Goyal S, Chauhan K. A review on analytical method development and validation. International Journal of Applied Pharmaceutics. 2018 Nov 7;10(6):8-15. Blessy MR, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating studies of drugs—A review. Journal of pharmaceutical analysis. 2014 Jun 1;4(3):159-65. Venkataraman S, Manasa M. Forced degradation studies: Regulatory guidance, characterization of drugs, and their degradation products-a review. Drug Invention Today. 2018 Feb 1;10(2). C Kogawa A, RN Salgado H. Impurities and forced degradation studies: a review. Current Pharmaceutical Analysis. 2016 Feb 1;12(1):18-24. Godela R, Gummadi S, Pathak S, Pola KK, Yagnambhatla R. RP-HPLC–PDA Approach for Concurrent Analysis of Telmisartan and Azelnidipine in Bulk and Commercial Tablets. Chemistry Africa. 2023 Feb;6(1):393-403. Additional Declarations No competing interests reported. Supplementary Files floatimage1.png GRAPHICAL ABSTRACT Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Oct, 2024 Reviews received at journal 04 Oct, 2024 Reviews received at journal 03 Oct, 2024 Reviews received at journal 02 Oct, 2024 Reviewers agreed at journal 29 Sep, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviewers agreed at journal 26 Sep, 2024 Reviewers agreed at journal 16 Sep, 2024 Reviewers invited by journal 21 Aug, 2024 Editor assigned by journal 20 Aug, 2024 Submission checks completed at journal 19 Aug, 2024 First submitted to journal 10 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4892587","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":353963952,"identity":"4eeb3232-9309-4854-835c-755c040f307b","order_by":0,"name":"Krishnaphanisri Ponnekanti","email":"","orcid":"","institution":"Malla Reddy Institute of Pharmaceutical Science","correspondingAuthor":false,"prefix":"","firstName":"Krishnaphanisri","middleName":"","lastName":"Ponnekanti","suffix":""},{"id":353963953,"identity":"7296bd8b-eeb6-4030-9435-ebb1050e91fc","order_by":1,"name":"Ramreddy Godela","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYNACNmYwJcFQwCAHYhx4QLwWAwZjsJYEUrQkNoBY+LSYt59O+/ChzDqxgX/xwRsfDOzS54cdfgi0xU5OtwG7FpkzuZtnzjiXntgg8SzZcoZBcu7G22kGQC3JxmYHsGuRYMjdzMzbdhio5YyZNI8Bc+7G2QkgLQcSt+HSwv92M/NfmJY/BvXphrPTP+DXIgG0hRGkhb/HTJrB4HCCvHQOAVsk3m5m7DmXbtwmwZZs2WNw3HCDdE7BgQQDPH7hz93M8KPMWraf//DBGz8qquXlZ6dv/vChwk4OlxYYcGyTSICwDMAqDfArBwF7Bn6oofINhFWPglEwCkbByAIAGE9hIQKxjo8AAAAASUVORK5CYII=","orcid":"","institution":"GITAM Deemed to be University","correspondingAuthor":true,"prefix":"","firstName":"Ramreddy","middleName":"","lastName":"Godela","suffix":""}],"badges":[],"createdAt":"2024-08-10 16:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4892587/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4892587/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64610973,"identity":"0dd676eb-5767-4c61-a520-99970e4ec3a7","added_by":"auto","created_at":"2024-09-16 14:01:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":294972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical structures of PRB and ETB.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/f725fa5ed9e9cde7b0c9f0a6.png"},{"id":64610197,"identity":"89254ce3-8b50-4899-8e56-d78733dffd9e","added_by":"auto","created_at":"2024-09-16 13:53:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":141312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatogram consisting of PRB and ETB peaks at optimized conditions.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/aee0972cd177931144a1c395.png"},{"id":64610194,"identity":"560eaac6-01d7-4b9f-8b3b-fa01497e627c","added_by":"auto","created_at":"2024-09-16 13:53:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":179632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinearity Curves of PRB and ETB.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/6fe0bada1ca50b30bc9fa063.png"},{"id":64610196,"identity":"5d5ff14b-3623-4897-840d-1dcc7881ac46","added_by":"auto","created_at":"2024-09-16 13:53:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":317437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms representing the specificity of the method.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/c77c689ad3d20ace4a8c88eb.png"},{"id":64610193,"identity":"be1f3c53-5c97-4f68-b56c-7eb6133cd595","added_by":"auto","created_at":"2024-09-16 13:53:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":306173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms and percentage degradation of PRB and ETB at various FD conditions.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/6158fe6fa07083d265fd5b88.png"},{"id":64611359,"identity":"9e23789b-e740-4622-a38e-5fd91c413738","added_by":"auto","created_at":"2024-09-16 14:09:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2012043,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/d7158008-47fe-409d-a0c8-c599bf300e7e.pdf"},{"id":64610192,"identity":"f03c2a98-5f30-428f-accb-0e32e25ff521","added_by":"auto","created_at":"2024-09-16 13:53:13","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":626534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cstrong\u003eGRAPHICAL ABSTRACT\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4892587/v1/3106e57c131e3869532f8d57.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eConcurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003ePregabalin (PGB) is an anticonvulsant and analgesic agent [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is well tolerated for managing neuropathic pain and chronic pain conditions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It has a wide safety margin. PGB is a first-line or adjunctive therapy for many conditions, including generalized anxiety disorders, diabetic peripheral neuropathy, and partial seizures [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Etoricoxib (ETB) is an NSAID and COX-2 inhibitor [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This is primarily used to treat people with gout, osteoarthritis, rheumatoid arthritis, and spondylitis-related joint pain and swelling [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is also employed to lessen edema and stiffness in the joints [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA Fixed-Dose Combination (FDC) is a product that combines two or more active ingredients in a fixed ratio and is used for treatment by integrating drugs, devices, or biological products. Defined by the U.S. FDA, FDCs enhance compliance, effectiveness, and cost-effectiveness while reducing pill burden and side effects [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They are widely prescribed globally for managing complex diseases such as HIV/AIDS, diabetes, hypertension, tuberculosis, neuropathic pain, asthma, and malaria. This approach streamlines treatment regimens, making them more efficient and patient-friendly. The chemistry of the PRB and ETB is illustrated in Fig.\u0026nbsp;1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The combination of PRB and ETB provides good therapeutic benefits compared to a single regimen treatment in pinched nerves with muscle weakness [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalytical methods are essential for accurately measuring drug amounts and purity, ensuring they are safe and effective. They check the quality of medicines, find impurities, and meet regulatory standards. These methods help develop new drugs by providing essential data. They also monitor drug levels in patients for proper dosing and fewer side effects. Overall, they ensure that medicines work correctly and safely.\u003c/p\u003e \u003cp\u003eAs of now, numerous UV techniques, chromatographic methods, and hyphenated methods like LC-MS and LC-MS/MS have been reported for PGB as a single drug moiety or in combination with other drugs [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Similarly, ETB possesses different analytical methods for identification and estimation [\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Few HPLC methods were reported in the literature for simultaneous estimation of PRB and ETB [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the reported methods, only some possess longer ETB retention time [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Few methods need stability-indicating properties [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. UPLC method is superior to HPLC in terms of lower RT, improved peak resolution, efficiency of peaks, and less solvent consumption due to less particle size of stationary phase and high-pressure pumps [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003ePRB and ETB in pure form were purchased from Spectrum Pharma in Hyderabad. All analytical and HPLC grade solvents were purchased from a nearby Merck India Limited vendor. Waters UPLC (TUV detector and Empower-2 software) was used to develop the method. Water (Milli-Q) and a 1 mg sensitive balance (SCALETEC-SAB224CL) were also utilized.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Method development\u003c/h2\u003e \u003cp\u003eThe method development was done using a trial-and-error concept. Different polar solvents with different rations were used to optimize the method by considering system suitability as per ICH. The effective separation of PRB and ETB was achieved with HSS column C18 (150x2.1mm,1.8\u0026micro;m), 0.1% orthophosphoric acid: acetonitrile in 65:35 v/v) at a flow rate of 0.3mL/min, isocratic elution at 228nm, and a column temperature of 30\u003csup\u003e0\u003c/sup\u003eC. Equal volumes of acetonitrile and water were used as a diluent to prepare the working standard, sample, and forced degradation solution.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Preparation of standard solution\u003c/h2\u003e \u003cp\u003eAccurately transferred about 75mg of PRB and 60mg of ETB pure powders into a volumetric flask measuring 100 mL to obtain750 \u0026micro;g/mL and 600 \u0026micro;g/mL for PRB and ETB. 1mL of the resultant solution was further made to 10 mL with diluent in a 10 mL calibrated volumetric flask to produce concentrations of 75 \u0026micro;g/mL and 60 \u0026micro;g/mL of PRB and ETB, respectively, which is referred to as 100% level concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Preparation of sample solution\u003c/h2\u003e \u003cp\u003eThe tablet powder equivalent 75 mg of PRB and 60 mg of ETB pure powders into a volumetric flask measuring 100 mL to obtain750 \u0026micro;g/mL and 600 \u0026micro;g/mL for PRB and ETB. 1mL of the resultant solution was further made to 10 mL with diluent in a 10 mL calibrated volumetric flask to produce concentrations of 75\u0026micro;g/mL and 60 \u0026micro;g/mL of PRB and ETB, respectively. The sample solution was filtered through the 0.45\u0026micro; filter to exclude the particulate matter.\u003c/p\u003e \u003cp\u003eMethod development\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Method validation\u003c/h2\u003e \u003cp\u003eThe validation of the method has been performed with the provision of ICHQ2R1 guidelines [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 System suitability\u003c/h2\u003e \u003cp\u003eSix replicate injections of a standard solution consisting of PRB (75\u0026micro;g/ mL) and ETB (60 \u0026micro;g/mL) were analyzed by the UPLC system with optimized conditions. The ICH recommended system suitability parameters, including theoretical plates, tailing factor, %RSD, and resolution, were determined to ensure the method's suitability toward the UPLC system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Linearity\u003c/h2\u003e \u003cp\u003eThe Linearity of the proposed method was established for PRB and ETB in the range of 18.75\u0026micro;g/mL to 112.5\u0026micro;g and 15\u0026micro;g/mL to 90\u0026micro;g/mL, respectively. A linear curve between concentration and peak area is used to get linear regression data to assess the slope, intercept, and regression coefficient(R\u003csup\u003e2\u003c/sup\u003e). The concentrations of about 18.75, 37.5,56.25,75,93.75 and 112.5\u0026micro;g/mL of PRB and 15,30,45,60,75 and 90\u0026micro;g/mL of ETB were analyzed in triplicate by the proposed approach to ensure the linearity range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Accuracy\u003c/h2\u003e \u003cp\u003eThe proposed UPLC method\u0026rsquo;s accuracy was ascertained by the standard addition method, where different levels (50%, 100%, and 150%) of standard solutions were separately spiked to a known amount of sample solution. The average (n\u0026thinsp;=\u0026thinsp;3) percentage recovery of various levels of standard solution from different spiked solutions was calculated from the recorded chromatograms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Precision\u003c/h2\u003e \u003cp\u003ePrecision is referred to as the reliability of the observed responses of a homogeneous sample over numerous replications. In most cases, Precision should be done on the same day (intraday) and distinct days(inter-day) as per ICH recommendations. Six repetitive injections of standard solution in a day and three repetitions per day for three consecutive days were analyzed to perform both intraday and inter-day precision. The %RSD was calculated for the peak areas that were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Robustness\u003c/h2\u003e \u003cp\u003eThe ability of the approach to withstand altering the parameters of the method in the analysis of the intended analyte without any changes in the analytical results. In the present approach, deliberate changes have been made to the mobile phase (\u0026plusmn;\u0026thinsp;1mL), column temperature (\u0026plusmn;\u0026thinsp;5\u003csup\u003eo\u003c/sup\u003eC), and flow rate (\u0026plusmn;\u0026thinsp;0.1mL/min). The system suitability parameters of chromatograms were obtained for standard solutions by injecting each altered parameter in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6 Specificity\u003c/h2\u003e \u003cp\u003eThe method's capacity to recognize the selected drug in the presence of other substances is referred to as specificity. Individual injections of 1\u0026micro;L of prepared blank solution, working standard solution, sample solution, and forced degradation solution were injected to detect any interference with the peaks of PRB and ETB from other peaks in the acquired chromatograms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.2.7 Sensitivity\u003c/h2\u003e \u003cp\u003eSensitivity in terms of Limit of Detection (LOD) and Limit of quantification (LOQ) was assessed by the standard deviation (SD) method, where the SD of the intercept and average slope of the linear curve (n\u0026thinsp;=\u0026thinsp;3) were considered.\u003c/p\u003e \u003cp\u003eLOD\u0026thinsp;=\u0026thinsp;3.3 σ/S\u003c/p\u003e \u003cp\u003eLOQ\u0026thinsp;=\u0026thinsp;10 σ/S\u003c/p\u003e \u003cp\u003eσ- SD of intercept of the linear plot, S- the average slope of the linear plot (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Forced degradation (FD) studies\u003c/h2\u003e \u003cp\u003eFD studies HPLC are intended to develop the stability-indicating method where stability and degradation of the intended analyte can be determined [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These FD studies help to assess the degradation behaviour and storage conditions to ensure drug safety and efficacy as per ICH requirements. These studies used the ICH Q1A(R2) and Q1B provisions [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe stability of PRB and ETB was examined under various FD conditions. In acid hydrolysis, 1mL of 2N HCl was mixed with 1mL of the standard stock solution, refluxed for 30 minutes at 60\u0026deg;C, and neutralized with the same strength NaOH. The resultant solution was diluted in such a way as to attain 75\u0026micro;g/mL and 60\u0026micro;g/mL of PRB and ETB, respectively. Base hydrolysis shadowed a similar process with 2N NaOH. Similarly, 20% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used in oxidative degradation. All three samples were kept aside for 48 hr. while thermal degradation involved exposing the stock solution for 72 hr at 105\u0026deg;C/75% RH. Photostability was determined by exposing the standard stock solution to UV light in the dark control for 7 days. In neutral degradation, equal portions of HPLC-grade water and standard stock solution were mixed uniformly. In each case, further dilution was done to get a solution of 75\u0026micro;g/mL of PRB and 60\u0026micro;g/mL of ETB. Each solution was analyzed in triplicate to determine the percentage degradation of PRB and ETB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Assay\u003c/h2\u003e \u003cp\u003eThe % purity of the commercial formulation can be assessed using an assay. It was carried out by injecting standard and sample solutions containing 75\u0026micro;g/mL of PRB and 60\u0026micro;g/mL of ETB, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Optimized method\u003c/h2\u003e\n \u003cp\u003eThe method development starts with initial conditions such as HSS column C18 (150x2.1mm,1.8\u0026micro;m), methanol: water in 50:50 v/v) at a flow rate of 0.3mL/min, isocratic elution at 228nm, and a column temperature of 30\u003csup\u003e0\u003c/sup\u003eC. A broad peak with a low plate count was observed for ETB. A further modification was made by replacing water in the state mobile phase with KH2PO4, which produced broad peaks for both PRB and ETB. Finally, a mobile phase of 0.1% OPA and acetonitrile (65:35 v/v) provided sharp and efficient peaks with plate counts of more than 2000. The retention time of PRB and ETB was noticed at 1.56 and 2.01 minutes, with good resolution and system suitability with the developed approach (Figure-2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Method validation\u003c/h2\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1 System suitability\u003c/h2\u003e\n \u003cp\u003eThe six replicate injections of the standard solution (PRB \u0026minus;\u0026thinsp;75\u0026micro;g/mL and ETB \u0026minus;\u0026thinsp;60 \u0026micro;g/mL) resulted in an average USP plate count more fabulous than 2000 (i.e., 2980) and a tailing factor of 2 or less (i.e., 1.4). The % RSD of peak response for PRB and ETB is two or less(i.e.,0.30). The mean resolution between PRB and ETB in the chromatographic peaks is 6.2, more significant than two. All the system suitability parameters results obeyed the ICH recommended limits (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of system suitability of the developed RP-UPLC method.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDrug name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStatistical parameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003cp\u003e(Min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak response\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUSP Plates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTailing factor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResolution\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean (n\u0026thinsp;=\u0026thinsp;6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e818272.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2353.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6765.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e109.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eETB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean (n\u0026thinsp;=\u0026thinsp;6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e785714.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2814.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2403.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2 Linearity\u003c/h2\u003e\n \u003cp\u003eThe developed method has shown significant linearity with an R\u0026sup2; value of 0.999 for the concentration ranges of PRB (18.75\u0026micro;g/mL to 112.5\u0026micro;g/mL) and ETB (15\u0026micro;g/mL to 90\u0026micro;g/mL)). These results reveal that the established method\u0026apos;s linearity aligns with ICH recommendations. The obtained chromatogram showing this linearity is shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. 3.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of linearity of PRB and ETB by developed method.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePRB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eETB\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak area\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak area\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e203260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e201015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e416912\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e610155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e622526\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e820299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e826289\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1020461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1031996\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1230021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1235425\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e(Correlation coefficient) R\u0026sup2;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.999\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.999\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.3 Accuracy\u003c/h2\u003e\n \u003cp\u003eThe average percentage recovery of PRB and ETB in 50%, 100%, and 150% levels of spiked solutions was calculated to be in the range of 99.0\u0026ndash;99.845% (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These results were within 100% \u0026plusmn; 2 limits as per ICH recommendations.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of percentage recovery of PRB and ETB.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDrug name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAmount of standard solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e*% Recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdded (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRecovered (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eETB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*Mean of three determinations\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.4 Precision\u003c/h2\u003e\n \u003cp\u003eIntraday and inter-day precision found that the computed % RSD values of the peak area responses standard solution of PRB and ETB in both intraday and inter-day precision were observed to be in the range of 0.27 to 0.55 (Table-4). The %RSD value less than or equal to two emphasizes the considerable precision of the established method.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eIntraday and inter-day results of PRB and ETB in standard solution.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eType of precision\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePeak area\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePRB\u003c/p\u003e\n \u003cp\u003e75 (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eETB\u003c/p\u003e\n \u003cp\u003e60 (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eIntraday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e826041.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2274.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e784085.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2274.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e%RSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eInter-day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e817685.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4549.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e785597.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3624.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e%RSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.5 Robustness\u003c/h2\u003e\n \u003cp\u003eThe slight and intentional alterations in the mobile phase, flow rate, and column temperature could not affect the method\u0026apos;s performance. The results of the obtained system suitability parameters complied with ICH guidelines (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), confirming the developed method\u0026apos;s robustness.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSystem suitability parameter data of PRB and ETB in the method\u0026apos;s robustness.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eVariation of Parameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ePregabalin(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak Area (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e%RSD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlate count\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTailing Factor\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMobile phase ratio (\u0026plusmn;\u0026thinsp;1mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66:34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e820830\u0026thinsp;\u0026plusmn;\u0026thinsp;6509.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64:36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e815294\u0026thinsp;\u0026plusmn;\u0026thinsp;4901.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow rate (\u0026plusmn;\u0026thinsp;0.1mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e816438\u0026thinsp;\u0026plusmn;\u0026thinsp;3517.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2443\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e816820\u0026thinsp;\u0026plusmn;\u0026thinsp;3868.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature (\u0026plusmn;\u0026thinsp;5\u0026ordm;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e818876\u0026thinsp;\u0026plusmn;\u0026thinsp;5841.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e816538\u0026thinsp;\u0026plusmn;\u0026thinsp;6349.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eEtoricoxib (n\u0026thinsp;=\u0026thinsp;6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMobile phase ratio (\u0026plusmn;\u0026thinsp;1mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66:34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e819353\u0026thinsp;\u0026plusmn;\u0026thinsp;4884.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64:36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e820997\u0026thinsp;\u0026plusmn;\u0026thinsp;5994.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow rate (\u0026plusmn;\u0026thinsp;0.1mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e820276\u0026thinsp;\u0026plusmn;\u0026thinsp;5772.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e823831\u0026thinsp;\u0026plusmn;\u0026thinsp;4652.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature (\u0026plusmn;\u0026thinsp;5\u0026ordm;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e821884\u0026thinsp;\u0026plusmn;\u0026thinsp;8372.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e813344\u0026thinsp;\u0026plusmn;\u0026thinsp;2297.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2829\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.6 Sensitivity\u003c/h2\u003e\n \u003cp\u003eThe LOD and LOQ of PRB and ETB were computed to be 0.07 \u0026micro;g/mL, 0.21 \u0026micro;g/mL, and 0.01\u0026micro;g/mL, 0.04 \u0026micro;g/mL, respectively, by standard deviation method.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.7 Specificity\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eNo interference from the placebo, blank, and degradant solution was found at the Pregabalin and Etoricoxib retention times (Fig. 4). It shows that the method is specific for estimating PRB and ETB under the stated method conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Forced degradation studies\u003c/h2\u003e\n \u003cp\u003eThe percentage degradation of PRB and ETB in degradation solutions was determined by comparing the peak responses of both analytes in freshly prepared standard solution with that of forced degradation solutions. The percentage degradation of PRB is higher in acidic conditions and very low in neutral degradation studies. Similarly, ETB has shown higher degradation in photolytic conditions and very low degradation in neural conditions. The above results significantly confirm the susceptibility of PRB and ETB toward acidic and photolytic environments, respectively (Fig. 5). The degradation of PRB and ETB in stated degradation conditions was identified by the developed approach regarding reduced peak response, and degradant peaks ensure the stability indicating feature of the established RP-UPLC method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Assay\u003c/h2\u003e\n \u003cp\u003eThe percentage purity of PRB and ETB in the marketed tablet was determined to be 99.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52% and 99.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61%, respectively (Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePercentage purity of PRB and ETB in commercial tablet.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDrug name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003cp\u003e(Min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAreas\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTailing factor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlate\u003c/p\u003e\n \u003cp\u003ecount\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAssay\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003cp\u003e(%w/w)\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePRB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStandard\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e817249\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e99.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e818637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2355\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eETB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStandard\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e787484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e99.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e789479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2877\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe developed UPLC method exerts advantages over the reported methods. No single UPLC method is available in the literature for determining PRB and ETB in combined dosage forms. In the reported HPLC methods, longer RT is needed, and ETB is needed more than 5 minutes [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. The RT of PRB and ETB in the current method is 1.5 and 2.0 min in that order. The LOD and LOQ values of the current approach were highly superior to the existing methods [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Stability-indicating features for HPLC or UPLC is highly significant in current scenarios in analytical methods to adopt the methods in regular analysis. All the reported methods lacked stability, indicating property, compared to the current method [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. The stated validation forced degradation studies results of the current method were aligned with the recommendations of ICH guidelines. The above results emphasize the developed approach\u0026apos;s efficacy, compatibility, and economic aspects.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eAn economical, robust, sensitive, and specific reverse-phase liquid chromatography approach was established, offering improved sensitivity and sample analysis time. The developed method was validated and verified under varied stress conditions, ensuring stability-indicating property and specificity towards PRB and ETB. This approach magnificently distinguishes PRB and ETB, along with their potential degradants, with high resolution. The shorter elution period and superior sensitivity of both analytes with this method are appropriate for regular analysis of PRB and ETB, confirming reliable results and effective separation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFUTURE PROSPECTIVE RESEARCH\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the current research study, the UPLC stability-indicating method was established. This method is highly effective in routinely analyzing the mentioned drugs in manufacturing units' bulk and dosage forms. To know the nature of degradants and impurities, further investigation of pharmaceuticals should be conducted using advanced hyphenated techniques like LC-NMR and LC-MS/MS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eETB-\u003c/strong\u003e Pregabalin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePRB-\u003c/strong\u003e Etoricoxib\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFD-\u0026nbsp;\u003c/strong\u003eForced Degradation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICH-\u0026nbsp;\u003c/strong\u003eInternational Committee for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRP-\u0026nbsp;\u003c/strong\u003eHPLC- Reverse Phase High-Performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLOD:\u0026nbsp;\u003c/strong\u003eLimit of Detection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLOQ:\u0026nbsp;\u003c/strong\u003eLimit of Quantification\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRSD:\u0026nbsp;\u003c/strong\u003eRelative Standard Deviation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSD:\u0026nbsp;\u003c/strong\u003eStandard Deviation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT:\u003c/strong\u003e Retention Time\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors contributed equally to the design and frame of the work, acquisition, interpretation of data, and manuscript preparation; all authors have read the prepared manuscript and approved it for\u0026nbsp;publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHUMAN AND ANIMAL RIGHTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and supportive information are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e The authors are thankful to the GITAM School of Pharmacy, GITAM Deemed to be University, and the Malla Reddy Institute of Pharmaceutical Sciences.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eToth C. Pregabalin: latest safety evidence and clinical implications for the management of neuropathic pain. Therapeutic advances in drug safety. 2014 Feb;5(1):38-56.\u003c/li\u003e\n \u003cli\u003eBen‐Menachem E. Pregabalin pharmacology and its relevance to clinical practice. Epilepsia. 2004 Aug;45:13-8.\u003c/li\u003e\n \u003cli\u003eKavoussi R. Pregabalin: from molecule to medicine. European Neuropsychopharmacology. 2006 Jul 1;16:S128-33.\u003c/li\u003e\n \u003cli\u003eDworkin RH, Kirkpatrick P. Pregabalin. Nature reviews Drug discovery. 2005 Jun 1;4(6):455-7.\u003c/li\u003e\n \u003cli\u003eLauria-Horner BA, Pohl RB. Pregabalin: a new anxiolytic. Expert opinion on investigational drugs. 2003 Apr 1;12(4):663-72.\u003c/li\u003e\n \u003cli\u003eShneker BF, McAuley JW. Pregabalin: a new neuromodulator with broad therapeutic indications. Annals of Pharmacotherapy. 2005 Dec;39(12):2029-37.\u003c/li\u003e\n \u003cli\u003eCochrane DJ, Jarvis B, Keating GM. Etoricoxib. Drugs. 2002 Dec;62:2637-51.\u003c/li\u003e\n \u003cli\u003eTakemoto JK, Reynolds JK, Remsberg CM, Vega-Villa KR, Davies NM. Clinical pharmacokinetic and pharmacodynamic profile of etoricoxib. Clinical pharmacokinetics. 2008 Nov;47:703-20.\u003c/li\u003e\n \u003cli\u003eDallob A, Hawkey CJ, Greenberg H, Wight N, De Schepper P, Waldman S, Wong P, DeTora L, Gertz B, Agrawal N, Wagner J. Characterization of etoricoxib, a novel, selective COX‐2 inhibitor. The Journal of Clinical Pharmacology. 2003 Jun;43(6):573-85.\u003c/li\u003e\n \u003cli\u003eMartina SD, Vesta KS, Ripley TL. Etoricoxib: a highly selective COX-2 inhibitor. Annals of Pharmacotherapy. 2005 May;39(5):854-62.\u003c/li\u003e\n \u003cli\u003ePatrignani P, Capone ML, Tacconelli S. Clinical pharmacology of etoricoxib: a novel selectiveCOX2 inhibitor. Expert Opinion on Pharmacotherapy. 2003 Feb 1;4(2):265-84.\u003c/li\u003e\n \u003cli\u003eKim DW, Weon KY. Pharmaceutical application and development of fixed-dose combination: Dosage form review. Journal of Pharmaceutical Investigation. 2021 Sep;51:555-70.\u003c/li\u003e\n \u003cli\u003ePubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 5486971, Pregabalin; [cited 2024 July 30]. Available from:\u0026nbsp;\u003ca href=\"https://pubchem.ncbi.nlm.nih.gov/compound/Pregabalin\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/Pregabalin\u003c/a\u003e\u003c/li\u003e\n \u003cli\u003ePubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 5486971, Pregabalin; [cited 2024 July 30]. Available from:\u0026nbsp;\u003ca href=\"https://pubchem.ncbi.nlm.nih.gov/compound/Pregabalin\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/Pregabalin\u003c/a\u003e\u003c/li\u003e\n \u003cli\u003eYeole AB, Sree Ranga Lakshmi G, Selvakumar CJ, Goni VG, Nawal CL, Valya BJ et al., Efficacy and Safety of Pregabalin Prolonged Release-Etoricoxib Combination Compared to Etoricoxib for Chronic Low Back Pain: Phase\u0026nbsp;3, Randomized Study. Pain Ther. 2022 Dec;11(4):1451-1469.\u003c/li\u003e\n \u003cli\u003eKasawar GB, Farooqui MN. Development and validation of HPLC method for the determination of pregabalin in capsules. Indian journal of pharmaceutical sciences. 2010 Jul;72(4):517.\u003c/li\u003e\n \u003cli\u003ePingale P, Singasane T. Development and validation of HPLC method for the determination of pregabalin in bulk and in pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2012;5(6):829-33.\u003c/li\u003e\n \u003cli\u003eMartinc B, Ro\u0026scaron;kar R, Grabnar I, Vovk T. Simultaneous determination of gabapentin, pregabalin, vigabatrin, and topiramate in plasma by HPLC with fluorescence detection. Journal of Chromatography B. 2014 Jul 1;962:82-8.\u003c/li\u003e\n \u003cli\u003eArayne MS, Shahnaz H, Ali A, Sultana N. Monitoring of Pregabalin in pharmaceutical formulations and human serum using UV and RP-HPLC techniques: Application to dissolution test method. Pharm Anal Acta. 2014;5(287):2.\u003c/li\u003e\n \u003cli\u003eRadwan RA, Abouzied NF, Badway FA, HA H, Hassan AM. New Method Development for Pregabalin Detection in Human Plasma by HPLCDAD. J. Pharm. Appl. Chem. 2020;6(3):61-.\u003c/li\u003e\n \u003cli\u003eBhatt KK, Patelia EM, Mori A. Simultaneous estimation of pregabalin and methylcobalamine in pharmaceutical formulation by RP-HPLC method. Journal of Analytical and Bioanalytical Techniques. 2013;4(1):1-4.\u003c/li\u003e\n \u003cli\u003eKannapan N, Nayak SP, Venkatachalam T, Prabhakaran V. Analytical RP-HPLC method for development and validation of pregabalin and methylcobalamine in combined capsule formulation.\u003c/li\u003e\n \u003cli\u003eKavitha MP, Rajasekhar A. A validated HPLC method for the analysis of pregabalin and methylcobalamin in bulk and pharmaceutical formulation. Pharmacie Globale. 2013 Jul 1;4(7):1.\u003c/li\u003e\n \u003cli\u003ePatel HM, Suhagia BN, Shah SA, Rathod IS. Determination of Etoricoxib in Pharmaceutical Formulations by HPLC Method. Indian Journal of Pharmaceutical Sciences. 2007 Sep 1;69(5).\u003c/li\u003e\n \u003cli\u003eTopalli S, Chandrashekhar TG, Annapurna MM. Validated RP‐HPLC Method for the Assay of Etoricoxib (A Non‐Steroidal Anti‐Inflammatory Drug) in Pharmaceutical Dosage Forms. Journal of Chemistry. 2012;9(2):832-8.\u003c/li\u003e\n \u003cli\u003eHartman R, Abrahim A, Clausen A, Mao B, Crocker LS, Ge Z. Development and validation of an HPLC method for the impurity and quantitative analysis of etoricoxib. Journal of liquid chromatography \u0026amp; related technologies. 2003 Aug 1;26(15):2551-66.\u003c/li\u003e\n \u003cli\u003ePattan SR, Jamdar SG, Godge RK, Dighe NS, Daithankar AV, Nirmal SA, Pai MG. RP-HPLC method for simultaneous estimation of paracetamol and etoricoxib from bulk and tablets. Journal of Chemical and Pharmaceutical Research. 2009 Jan;1(1):329-35.\u003c/li\u003e\n \u003cli\u003eKumar S, Joshi A, Thakur RS, Pathak AK, Shah K. Simultaneous estimation of etoricoxib and thiocolchicoside by RP-HPLC method in combined dosage forms. Acta Poloniae Pharmaceutica. 2011 Nov 1;68(6):839-43.\u003c/li\u003e\n \u003cli\u003eMandal U, Senthil Rajan D, Bose A, Gowda KV, Ghosh A, Pal TK. Development and Validation of an HPLC Method for Analysis of Etoricoxib in Human Plasma. Indian journal of pharmaceutical sciences. 2006 Jul 1;68(4).\u003c/li\u003e\n \u003cli\u003eAlzweiri M, Sallam M, Al-Zyoud W, Aiedeh K. Stability study of etoricoxib a selective cyclooxygenase-2 inhibitor by a new single and rapid reversed phase HPLC method. Symmetry. 2018 Jul 17;10(7):288.\u003c/li\u003e\n \u003cli\u003eKaranjkar MB. Development And Validation Of RP-HPLC For Simultaneous Estimation of Pregabalin and Etoricoxib in Pharmaceutical Tablet Dosage Form. IJPSR. 2021;13(4):872-9.\u003c/li\u003e\n \u003cli\u003eYeluri RR, Reddy BS, Kumari RR. Quantification of Pregabalin and Etoricoxib Combo in Tablets and Bulk with Developed RP-HPLC Method: Stability Indicating Feature Assessment. Journal of Advanced Scientific Research. 2022 May 30;13(04):31-6.\u003c/li\u003e\n \u003cli\u003eHanumanth V, Kumar PR, Sravya K, Padmavathi Y, Babu NR, Sowmya K. Simultaneous Estimation of Pregabalin and Etoricoxib in Bulk and Pharmaceutical Dosage Form by Using RP-HPLC Method. Journal of Drug Delivery and Therapeutics. 2023 Sep 15;13(9):20-7.\u003c/li\u003e\n \u003cli\u003eChaudhary A, Singh BK. Simultaneous Estimation of Pregabalin and Etoricoxib using Novel HPLC Method: An Application in Quantitative Analysis of Pharmaceutical Dosage Forms. Indian Journal of Pharmaceutical Education and Research. 2021 Jul 1;55:191.\u003c/li\u003e\n \u003cli\u003eNov\u0026aacute;kov\u0026aacute; L, Matysov\u0026aacute; L, Solich P. Advantages of application of UPLC in pharmaceutical analysis. Talanta. 2006 Jan 15;68(3):908-18.\u003c/li\u003e\n \u003cli\u003eRavisankar P, Navya CN, Pravallika D, Sri DN. A review on step-by-step analytical method validation. IOSR J Pharm. 2015 Oct;5(10):7-19.\u003c/li\u003e\n \u003cli\u003eSharma S, Goyal S, Chauhan K. A review on analytical method development and validation. International Journal of Applied Pharmaceutics. 2018 Nov 7;10(6):8-15.\u003c/li\u003e\n \u003cli\u003eBlessy MR, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating studies of drugs\u0026mdash;A review. Journal of pharmaceutical analysis. 2014 Jun 1;4(3):159-65.\u003c/li\u003e\n \u003cli\u003eVenkataraman S, Manasa M. Forced degradation studies: Regulatory guidance, characterization of drugs, and their degradation products-a review. Drug Invention Today. 2018 Feb 1;10(2).\u003c/li\u003e\n \u003cli\u003eC Kogawa A, RN Salgado H. Impurities and forced degradation studies: a review. Current Pharmaceutical Analysis. 2016 Feb 1;12(1):18-24.\u003c/li\u003e\n \u003cli\u003eGodela R, Gummadi S, Pathak S, Pola KK, Yagnambhatla R. RP-HPLC\u0026ndash;PDA Approach for Concurrent Analysis of Telmisartan and Azelnidipine in Bulk and Commercial Tablets. Chemistry Africa. 2023 Feb;6(1):393-403.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Applied Sciences](https://link.springer.com/journal/42452)","snPcode":"42452","submissionUrl":"https://submission.springernature.com/new-submission/42452/3","title":"Discover Applied Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pregabalin, Etoricoxib, RP-UPLC, isocratic elution, Stability indicating, C18 column","lastPublishedDoi":"10.21203/rs.3.rs-4892587/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4892587/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present research aims to establish new stability indicating RP-UPLC method for concurrently estimating Pregabalin and Etoricoxib in blended powder and their combined tablet formulation. The effective separation of Pregabalin and Etoricoxib achieved with HSS column C18 (150x2.1mm,1.8µm), 0.1% orthophosphoric acid: acetonitrile in 65:35 v/v) at a flow rate of 0.3mL/min, and isocratic elution at 228nm. The elution of PRB and ETB was noticed at 1.56 and 2.01 minutes, with good resolution and system suitability with the developed approach. PRB and ETB have shown linear responses from 18.75 to 112.5µg/mL and 15 to 90µg/mL, respectively. The range of the % RSD for intraday and inter-day precision was 0.33 to 0.81. The LOD and LOQ of Pregabalin and Etoricoxib were computed to be 0.07 µg/mL, 0.21 µg/mL, and 0.01µg/mL, 0.04 µg/mL, respectively, by standard deviation method. The validation method was carried out using ICH standards. The stability- indicating the feature of the method was confirmed by the forced degradation studies where degradants generated by stress testing were clearly distinguished from the peaks of analytes. The shorter elution period and superior sensitivity of both analytes with this method are appropriate for regular analysis of Pregabalin and Etoricoxib.\u003c/p\u003e","manuscriptTitle":"Concurrent Estimation of Pregabalin and Etoricoxib by New Stability Indicating Rp-uplc Approach- Application in Assay of Commercial Tablets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 13:53:08","doi":"10.21203/rs.3.rs-4892587/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-09T05:02:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-04T08:00:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-04T03:13:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-02T18:38:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172599665730933085023548603066934119502","date":"2024-09-30T03:37:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76230680734474776942785465051018996549","date":"2024-09-27T09:45:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338545375835675445570589897412772145184","date":"2024-09-26T16:28:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150103414817410874585538238289829463690","date":"2024-09-16T08:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-21T04:16:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-20T15:03:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-19T07:46:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Applied Sciences","date":"2024-08-10T16:45:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Applied Sciences](https://link.springer.com/journal/42452)","snPcode":"42452","submissionUrl":"https://submission.springernature.com/new-submission/42452/3","title":"Discover Applied Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b201412f-728c-435b-9000-d98ad7d25d26","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T10:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 13:53:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4892587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4892587","identity":"rs-4892587","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}