Novel Chromatographic Method Development and Validation for Nitroglycerin Determination in Semi-Solid Dosage Forms by RP-HPLC

Novel Chromatographic Method Development and Validation for Nitroglycerin Determination in Semi-Solid Dosage Forms by RP-HPLC | 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 Method Article Novel Chromatographic Method Development and Validation for Nitroglycerin Determination in Semi-Solid Dosage Forms by RP-HPLC Pradip Patil, Bhavin Dhaduk, Pankajkumar Nariya, Mahesh Savant, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8723511/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 method was validated according to ICH Q2(R2) guidelines included the system suitability, specificity, linearity, accuracy, homogeneity, robustness and forced degradation. The report exhibits a robust and stability-indicating RP-HPLC method developed for the quantification of Nitroglycerin in 4 mg/g ointment formulations. Chromatographic separation was performed using an L1 packing C18 column (150 mm × 4.6 mm, 5 µm) with an isocratic mobile phase of water and methanol in a 60:40 (v/v) ratio. The flow rate was maintained at 1.0 mL/min, and detection was carried out using a UV detector set at 220 nm and run time was 15.0 min. It revealed excellent system suitability, specificity without interference from excipients, impurities, degradants, or matrix components and linearity over the range of 40–120 µg/mL. Percentage accuracy (recovery) values obtained for the NTG is from 99.3% to 100.1%. Photodiode array (3D-PDA) detector unveils homogeneity of the NTG peak under stress conditions confirmed uniform distribution across formulation layers. Robustness process confirmed method reliability, method transfer and reduced errors and reliability under varied conditions. Forced degradation study reveal specificity and its ability to distinguish NTG from its degradation products and metabolites, without interference under the various stress conditions such as acid and base hydrolysis, oxidation, thermal, humidity, and photolytic degradation. The assessment of greenness of analytical HPLC was carried out using Analytical Eco-Scale, AGREE, and GAPI. The Eco-Scale score of 87, the AGREE tool yielded a score of 0.63 and the GAPI pictogram revealed a balanced profile. Therefore, such worthwhile quantification study intended for regulatory compliance approach in the pharmaceutical and healthcare avenue. Nitroglycerin RP-HPLC Stress studies Validation Routine analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Nitro-glycerine (NTG), also known as glyceryl trinitrate, is a potent organic nitrate compound extensively used in the treatment of cardiovascular conditions, primarily angina pectoris, heart failure and anorectal disorders such as chronic anal fissures [ 1 – 3 ]. NTG functions as a potent vasodilator, exerting its therapeutic effect by releasing nitric oxide (NO), which relaxes vascular smooth muscle and reduces myocardial oxygen demand [ 4 ]. Among the various dosage forms, topical ointments (4 mg/gm) offer a controlled and sustained release of the active pharmaceutical ingredient through transdermal absorption, minimizing first-pass metabolism and ensuring steady plasma levels over time [ 5 ]. Due to its volatile and chemically unstable, NTG might be degrades into metabolites such as 1,2-diniroglycerin and 1,3-diniroglycerin when exposed to the heat, light or moisture. This degradation compromises therapeutic efficacy and raises safety concerns [ 6 – 9 ]. Therefore, it is crucial to develop a highly sensitive analytical method for the accurate quantification and characterization of NTG during manufacturing and product release to ensure the product's quality, safety, and efficacy throughout its shelf life. A review of the literature reveals that various analytical techniques such as UV spectroscopy, HPLC, LC-MS, and GC-MS have been reported for the analysis of NTG and its mono and dinitrate metabolite across diverse matrices, including bulk drugs, pharmaceutical formulations, and human plasma [ 8 , 10 – 17 ]. However, most existing methods were time consuming, lack sensitive and unable to distinguish the decomposition products. Additionally, primarily focus on quantifying NTG without adequately addressing the regulatory requirements for controlling mono and dinitrate metabolites in Nitro-glycerine commercial dosage form [ 18 ]. Currently, no suitable analytical method is available for the simultaneous and sensitive detection of NTG and its mono and dinitrate metabolites in accordance with regulatory expectations. To address this gap, we have developed a highly selective HPLC method that significantly improves sensitivity compared to existing techniques. This method is particularly valuable for monitoring NTG and its dinitrate metabolite. All critical performance parameters including specificity, recovery, reproducibility, and linearity have been successfully validated. The method serves as a robust and reliable tool for routine quality control analysis, ensuring both the safety and regulatory compliance of NTG containing pharmaceutical products. The chemical structures of NTG and its dinitrate metabolite are shown in Fig. 1 . Method detail Chemicals and Reagents All reagents and solvents used in this study were of analytical grade and were used without further purification. HPLC-grade methanol was procured from JT Baker, Mumbai, India. 30% hydrogen peroxide, sodium hydroxide and hydrochloric acid were purchased from Merck. Mumbai, India. Milli-Q water collected from the Milli-Q Plus water purification system (Millipore, Milford, MA, USA) was used throughout the analysis. An in-house working standard of NTG (potency 9.74%) and reference standards of its metabolites such as 1-mononitroglycerin (98.38%), 2-mononitroglycerin (95.44%), 1,2-dinitroglycerin (92.57%), and 1,3-dinitro glycerine (95.26%) were employed for method development, optimization, and validation. PTFE syringe (0.45 µm) filters were used for the filtration of the sample solutions. Preparation of analytical solutions Preparation of standard stock solution of NTG (800 µg/mL): Weight accurately 821.22 mg of NTG working standard to get effective 80 mg of pure NTG. Transfer the weighed material into 100 mL volumetric flask. Add about 70-80 mL of methanol and sonicate to dissolve. Dilute up to the mark with methanol and mix well. Preparation of standard test solution of NTG (80 µg/mL): Take 10 ml of standard stock solution of NTG into 100 ml volumetric flask and dilute up to the mark with methanol and mix well. Preparation of sample solution of NTG pharmaceutical dosage form (40 µg/mL): Accurately weigh and transfer ointment equivalent to 4 mg of NTG into a 100 mL stoppered glass flask. Add approximately 50 mL of diluent using a calibrated pipette. Place the flask in a water bath maintained at 50 ± 2 °C for 12 min , shaking the flask every 4 minutes to ensure uniform dispersion of the sample. After 12 min, shake the flask vigorously for 2 min to obtain a homogeneous viscous solution. Repeat the above process twice to ensure consistency. Transfer the final solution into a 20 mL glass syringe and filter through a 0.45 µm PVDF filter . Discard the initial 4 mL of the filtrate to saturate the filter. Homogeneity sample preparation: To assess homogeneity, NTG ointment was sampled from multiple-dose containers. Samples were collected from the initial, middle, and end portions of each of five different marketed containers. The same sample preparation procedure described above was applied to each portion. Instrumentations An HPLC system (Waters Alliance) comprising an integrated pump with inbuilt degasser, auto sampler, and column thermostat (Model: 2695 Separation Module), along with a variable UV wavelength detector (Model: 2489) and a photodiode array (PDA) detector (Model: 2998) from Waters Corporation (USA), was used for the detection of Nitro-glycerine (NTG). Data acquisition and processing (FR5) were carried out using Empower 3 software. The pH of the buffer solution was measured using a seven-excellence digital pH meter (Mettler Toledo, USA). Deaeration of the mobile phase was performed using a USB 6.5 L ultrasonic bath (PCi Analyst). Chromatographic condition The chromatographic separation was carried out using an -C18 column (150 mm × 4.6 mm, 5 µm particle size) L1- Octadecylsilane chemically bonded to porous or non-porous silica or ceramic microparticles, 1.5 to 10µm in diameter . The mobile phase consisted of purified water and methanol in a 60:40 (v/v) ratio, delivered in isocratic mode at a flow rate of 1.0 mL/min. The column was maintained at ambient temperature, and the injection volume was set to 20 µL. The total run time for each analysis was 15.0 min. The optimized chromatographic parameters used for the effective separation of NTG and its metabolites are summarized in Table 1 . Table :1 chromatographic parameter Parameters Condition Mode HPLC Column C18 4.6 X 150 mm , 5 µm (L1 packing ) Wavelength 220 nm Flow rate 1.0 ml/ min Injection volume 20 µl Column over temperature Ambient Run time 15 min the retention of Glyceryl Trinitite Peak system suitability : USP Tailing factor Not more than 2.0 USP Plate count Not less than 2000 % RSD of Five standards injection Not more than 2.0 % Force degradation Forced degradation studies were performed on Nitro-glycerine (NTG) ointment at a concentration equivalent to 4 mg/gram to evaluate the stability and specificity of the developed HPLC method. Samples were subjected to various stress conditions, including acid hydrolysis, base hydrolysis, oxidation, thermal, humidity, and photolytic degradation. Placebo and blank were also evaluated to confirm no interference at the retention time of NTG and known impurities. After the designated exposure periods, samples were neutralized as necessary, diluted, and analyzed using the developed HPLC method with PDA detection. Peak purity was assessed using 3D-PDA data, where the purity angle was compared against the purity threshold to evaluate the homogeneity of the NTG peak. Results Method development and optimization Before initiating the development of the LC method for the quantification of NTG in topical ointment formulations, essential information about the compound was gathered from literature and experimental data. NTG is a clear, colourless to slightly yellow liquid with a melting point of approximately 14 °C and a boiling point near to 50 °C. It is miscible with acetone and anhydrous ethanol, and possesses a pKa of 5.6, while exhibiting a neutral pH of 7. 0 [17]. Due to the polar nature, reverse phase chromatography was chosen for method development. The primary objective was to develop a selective and robust LC method that could effectively separate and quantify NTG from formulation excipients. A major challenge was to achieve appropriate retention with symmetrical peak shape and absence of placebo interference. Considering these requirements, multiple chromatographic trials were carried out by varying mobile phase composition, column type, and system parameters to determine suitable separation conditions. The wavelength of 220 nm was selected for detection based on UV spectral scan of a standard solution (80 µg/mL), which demonstrated optimal absorbance and peak purity. In the initial trial, a mobile phase consisting of methanol:water (50:50, v/v) was tested on an Inertsil C18 column. A distinct peak was obtained at 10.5 min, failed to comply with USP system suitability criteria. In the second trial, a Hypersil BDS C18 column was used with a modified mobile phase ratio of methanol:water (60:40, v/v) and a flow rate of 1.3 mL/min. The result showed a distinct peak of NTG with retention time of 7.7 min; however, system suitability parameters were not met. A third trial using an Alpha C18 column (4.6 × 150 mm, 5 µm) under similar conditions yielded a retention time of 5.5 min, but failed to comply with USP system suitability criteria. To overcome these limitations, further optimization was performed using an C18 column (4.6 × 150 mm, 5 µm) L1 Packing with methanol:water (60:40, v/v) as the mobile phase, a flow rate of 1.0 mL/min, injection volume of 50 µL, and a column temperature maintained at 25 °C . The resulting chromatogram showed a well-resolved NTG peak at 5.5 minutes with excellent peak symmetry, and acceptable theoretical plate count and tailing factor. This optimized chromatographic condition was selected as the final method for further validation as per ICH guidelines. The finalized method demonstrated robustness, reproducibility, and compliance with all system suitability criteria, ensuring its suitability for routine quantification of NTG in pharmaceutical formulations. Method Validation The proposed method was validated for specificity, precision, accuracy, linearity, and limits of detection and quantification according ICH Q2 (R2) guidelines [19-22]. System suitability testing System suitability testing was conducted to confirm that the analytical system was fit for its intended purpose at the time of analysis. This evaluation involved six replicate injections (n = 6) of a NTG standard test solution at a concentration of 80 µg/ml. The average retention time for NTG was 5.71 min, with a % RSD of peak areas calculated at 0.43%. Since the % RSD was well within acceptable limits, the system was deemed suitable for analysis [23, 24]. A summary of these results is presented in Table 2. Table 2. Results of validation parameters: a) System suitability b) Specificity c) Linearity d) Recovery / accuracy e) Method Precision (Inter / Intra) Parameters Acceptable criteria Result System suitability (Mean ± RSD) (n =6) Retention time %RSD ≤ 2.0% 5.41 ± 0.43 % USP tailing factor ≤ 2.0 1.2 ± 3.15 % USP plate count ≥ 2000 7329 ± 0.67 % Standard area %RSD ≤ 2.0% 17702 ± 0.67 % Specificity No interference at retention time of NTG or known impurities No interference Linearity (40 - 120 µg/ml) Regression equation Line equation should show good fit 3E-08x + 0.025 Slope Positive slope indicating sensitivity 3E-08x Correlation coefficient (r 2 ) ≥ 0.999 0.9993 % Recovery (Mean ± RSD) Level-1 (50%) 98.0% – 102.0%, RSD ≤ 2.0% 100.1 ± 0.20 % Level-2 (100%) 99.3 ± 0.32 % Level-3 (150%) 99.9 ± 0.07 % Precision (Mean ± RSD) Intraday (n = 6) %RSD ≤ 2.0% 1902837.17 ± 0.87 % Inter day (n = 6) 1911050.67 ± 0.35 % Note: R 2 = determination of coefficients, RSD = Relative standard deviation. Specificity The specificity of the method was evaluated by assessing potential interfering components in the HPLC spectrogram. A 80 µL solution of blank, standard test solutions of 1-Mononitroglycerin, 2-Mononitroglycerin, 1,2-Dinitroglycerin, and 1,3-Dinitroglycerin and NTG, and a spiked sample (containing both NTG and its metabolites) were injected separately into the HPLC system. The results demonstrated no interference from any interfering components, and all analyte peaks remained distinct and unaltered. The spectrogram obtained are shown in Figure 2 to 6. The results revealed that there was no interference of the NTG with its metabolites peaks and hence the specificity of the developed method was proven. Figure 2 to 5 – HPLC Assay Chromatogram : Linearity The linearity of the NTG was satisfactorily demonstrated with a five-point calibration graph, ranging from the 50 to 150 % of the specification level (actual concentration: 0.04, 0.06, 0.08, 0.10, and 0.12, μg/mL). The slope, intercept and correlation coefficient values were derived from linear least-square regression analysis and the data is presented in Table 2 and figure 6. It reveals that an excellent correlation existed between the peak area concentrations of NTG. Accuracy The recovery testing involved spiking a known quantity of NTG with placebo at various levels (50% = 40 μg/mL, 100% = 80 μg/mL, and 150% = 120 μg/mL of the specification). Each concentration level was injected triplicate into the HPLC. The % recovery values obtained for the NTG ranged from 99.3% to 100.1%, which are within the acceptance criteria. The % RSD values of recoveries for the NTG ranged from 0.07 % to 0.32 % at 50 to 150 % levels. The % recovery results are shown in Table 2. Precision A precision study was conducted by injecting a standard test samples (n = 6) of NTG to evaluate intra and inter day precision. The % RSD obtained for intra, and inter day precision were 0.87, and 0.35 %, respectively. These results demonstrate that the method is sufficiently precise, with detailed precision data provided in Table 2 and Table 3 a and 3b. Table No 3a; Results of Inter and Intra days precision Interday Day 1 Day 2 Mean %RSD Injection 1 2 3 1 2 3 Area 1916781 1896798 1930011 1889865 1892314 1891254 1902837.17 0.873 Intraday Morning Evening %RSD Injection 1 2 3 1 2 3 Area 1910023 1916358 1910012 1912817 1901293 1921201 1911050.67 0.352 B ) Method precision and Homogeneity : Method precision was verified by analysing the six samples of the assay with bath analysis and Initial , middle and bottom in triplicate samples as per method and results are tabulated in Table 7 and 8 . Table No 3b: Results of Method precision and Homogeneity Sample No % Assay Nitroglycerin (Glyceryl Trinitite) Sample No. % Assay Nitroglycerin (Glyceryl Trinitite for homogeneity 1 99.8 Initial 1 100.1 2 99.1 2 99.7 3 99.4 3 99.2 4 99.5 Middle 1 98.9 5 98.8 2 99.5 6 100.2 3 99.5 Mean 99.5 End 1 97.9 % RSD 0.50 2 98.6 Limit % Assay Nitroglycerin (Glyceryl Trinitite) -90.0 to 105.0 % % RSD of 6 samples not More than 2.0 3 98.2 Mean 99.1 % RSD 0.73 Limit % Assay Nitroglycerin (Glyceryl Trinitite) -90.0 to 105.0 % % RSD of 9 samples not More than 2.0 Sample homogeneity Sample homogeneity of NTG ointment (4 mg/g) was confirmed by analyzing six assay samples taken from the beginning, middle, and end of a batch. The mean assay was 99.1% with a %RSD of 0.73%, indicating uniform API content throughout. Method precision and homogeneity data are shown in Table 4. Table 4: Method precision and sample homogeneity data of NTG ointment (4 mg/g) Sample No. % Assay – Method Precision Sampling Location % Assay – Sample Homogeneity 1 99.8 Initial 100.1 2 99.1 99.7 3 99.4 99.2 4 99.5 Middle 98.9 5 98.8 99.5 6 100.2 99.5 – – End 97.9 – – 98.6 – – 98.2 Mean 99.5 99.1 % RSD 0.50 0.73 Acceptance Criteria 90.0% – 105.0%, % RSD ≤ 2.0 90.0% – 105.0%, % RSD ≤ 2.0 Robustness The robustness of the HPLC method for Nitroglycerin (NTG) assay was assessed by introducing deliberate variations in flow rate, column temperature, detection wavelength, sample heating time and temperature, and mobile phase composition. As summarized in Table 5, all assay results remained within the acceptable range of 98.0 - 102.0 %, with %RSD values below 2.0%. System suitability parameters including USP tailing (≤ 2.0) and plate count ( ≥ 2500) were consistently met. Although a slight variation was noted under altered heating temperature conditions (absolute difference = 2.0 %), it remained within acceptable limits, confirming the method's robustness [25]. Table 5. Result of Robustness study Sr. No Change in parameters USP Tailing USP Plate count % Recovery Assay Mean % RSD Absolute % difference 1 Flow rate (ml/min) 0.8 1.2 7109 100.2 100.2 0.1 0.1 1.0 1.2 7341 100.3 1.2 1.2 7002 100.1 2 Column temperature (°C) 20.0 1.2 7198 99.9 100.0 0.1 0.2 25.0 1.1 7309 99.9 30.0 1.2 6912 100.1 3 Wavelength (nm) 216 1.2 7425 98.9 99.7 0.7 1.3 218 1.2 7387 100.2 222 1.2 7410 99.9 4 Sample heating time (min) 9.0 1.1 7413 98.9 99.5 0.5 1.0 12.0 1.3 7375 99.9 15.0 1.2 7376 99.6 5 Sample heating temperature (°C) 45.0 1.2 7402 96.9 98.7 1.7 2.0 50.0 1.2 7387 100.2 55.0 1.2 7365 98.9 6 Nobile phase composition - Methanol : Water (% v/v) 50:50 1.1 6895 100.1 100.1 0.2 0.3 60:40 1.1 7383 100.2 70:30 1.3 6843 99.9 Force degradation study NTG ointment samples were subjected to various stress conditions, including acid hydrolysis, base hydrolysis, oxidation, thermal, humidity, and photolytic degradation [26]. It was observed that NTG was stable under all tested stress conditions including acid, oxidative, thermal, photolytic, and humidity degradation, with minor degradation (<5%). Base hydrolysis resulted in moderate degradation (3.4 %), but peak purity remained acceptable, confirming the method's specificity and stability-indicating capability. No interfering peaks were observed in the blank or placebo chromatograms at the NTG retention time. The force degradation results are summarized in Table 6. Table 6: Force degradation results for NTG Ointment (4 mg/g) Sr. No Mode of Degradation Condition % Assay Absolute Degradation Purity Angle Purity Threshold Purity Flag 1 Control - 99.7 - 0.130 0.290 NO 2 Acid Hydrolysis 2 mL of 0.25 N HCl at 25 °C / 20 min 95.2 4.5 0.152 0.312 NO 3 Base Hydrolysis 2 mL of 0.01 N NaOH at 50 °C / 20 min 95.5 3.4 0.123 0.310 NO 4 Oxidative Degradation 2 mL of 30% H₂O₂, 25 °C / 24 hrs 97.7 2.0 0.129 0.345 NO 5 Humidity Degradation 25 °C / 90% RH for 7 days 99.2 0.5 0.135 0.299 NO 6 Photolytic Degradation 1.2 Million Lux Hours, 200 W·hr/m² 99.0 0.7 0.139 0.296 NO 7 Thermal Degradation 40 °C for 48 hrs 98.2 0.7 0.120 0.321 NO Application of the method for real sample analysis Three commercial-scale batches of NTG topical ointment (4 mg/g) were analyzed using the developed RP-HPLC method. Test sample solutions were prepared according to the validated procedure and subjected to chromatographic analysis. The results confirmed the presence of NTG at the labelled concentration in all three batches, with no detectable levels of mono or dinitrate impurities. To assess method specificity, known amounts of NTG mono- and dinitrate impurities were spiked into the test samples. The method successfully separated all analytes without interference from the formulation matrix. The spiked impurities were recovered within acceptable limits (98.5%–101.2%), confirming that method is suitable for routine quality control of bulk and finished products. The results are summarized in Table 2 and 3a and 3b . Comparison of the present method with the earlier reported methods The analytical performance of the proposed RP-HPLC method was evaluated and compared with previously reported techniques in terms of sensitivity, accuracy, specificity and precision run time, and selectivity (Table 2). Unlike earlier methods, which focused developed on quantification of NTG in different matrices, the present method is specifically developed for the identification and quantification of a NTG and its mono and dinitrate metabolites in 4 mg/g Topical Ointment, in accordance with ICH Q2(R2) guidelines. Moreover, none of the existing reported methods address this specific impurity. The proposed method offers shorter run time (15 min) and uses low environmental impact reagents, and demonstrates improved selectivity, high accuracy, and cost-effectiveness, making it valuable advancement for impurity control in pharmaceutical. Assessment of Greenness of Analytical HPLC The greenness of the developed RP-HPLC method for the quantification of NTG and its nitrate metabolites was evaluated using three complementary metrics: Analytical Eco-Scale , AGREE , and GAPI [27] . The Eco-Scale score of 87 Table S1 classifies the method as excellent in terms of greenness. The AGREE tool yielded a score of 0.63 , indicating good alignment with green analytical chemistry principles, particularly due to its low energy usage and direct, multi-analyte analysis. The GAPI pictogram revealed a balanced profile, with strengths in instrumentation and direct measurement but concerns due to solvent consumption and heating during sample preparation. Overall, the method qualifies as environmentally acceptable and suitable for routine analytical applications, with scope for further improvement by adopting greener solvents. The greenness scores for the proposed method were evaluated and presented in Table S2. Conclusion A robust, precise, and stability-indicating RP-HPLC method was successfully developed and validated for the quantification of NTG in 4 mg/g ointment formulations. The method employed a simple isocratic elution with water and methanol (60:40, v/v) on a C18 column, offering good resolution and a retention time of approximately 5.7 minutes. The method demonstrated excellent linearity, precision, and accuracy across the specified concentration range (40–120 µg/mL), with recovery rates between 99.3% and 100.1%. Forced degradation studies confirmed the method's specificity and its ability to distinguish NTG from its degradation products and metabolites, without interference. The peak purity analysis via PDA confirmed the homogeneity of the NTG peak under stress conditions. Additional validation parameters such as filter compatibility, solution and mobile phase stability, homogeneity, and robustness further confirmed the reliability and suitability of the method for routine quality control analysis. Overall, this validated method meets all the ICH Q2(R2) criteria and is well-suited for the routine release testing and stability studies of NTG ointment, ensuring compliance with regulatory expectations and safeguarding product quality and efficacy throughout its shelf life. Declarations Acknowledgment The author gratefully acknowledges Department of Chemical Sciences, Faculty of Science, Atmiya University for their generous support, valuable guidance, and for providing all the necessary facilities that contributed significantly to the successful completion of this work and Jegan Balan for analytical resources and execution help. Credit author statement Pradip Patil: Project administration, Methodology, Investigation, conducting the experiments, Bhavin Dhaduk: Analysis data acquisition, Writing. Pankajkumar Nariya and Shamkumar Deshmukh: Investigation, Methodology, Funding acquisitions. Mahesh Savant: Writing review and editing data acquisition. Funding This research was self-funded by the authors. No external financial support was received for the conduct of this study. Data Availability The data supporting the findings of this study are included within this paper. Ethics approval and consent to participate This study did not involve human participants or animals. Therefore, ethical approval was not required. This study did not involve human participants. Clinical trial number: Not applicable Consent for publication This study does not contain any individual person’s data. Competing interests The authors declare no competing interests. References Twiner MJ, Hennessy J, Wein R, Levy PD (2022) Nitroglycerin use in the emergency department: current perspectives. Open Access Emerg Med 14 :327–333. Yousif EM et al (2012) Formulation and clinical testing of glyceryl trinitrate ointment for treatment of anal fissures. Am J Pharmacol Toxicol. Lund JN, Scholefield JH (1997) A randomised, prospective, double-blind, placebo-controlled trial of glyceryl trinitrate ointment in treatment of anal fissure. Lancet 349 :11–14. Divakaran S, Loscalzo J (2017) The role of nitroglycerin and other nitrogen oxides in cardiovascular therapeutics. J Am Coll Cardiol. 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Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.jpg Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Mar, 2026 Reviews received at journal 09 Mar, 2026 Reviews received at journal 05 Mar, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviews received at journal 25 Feb, 2026 Reviewers agreed at journal 22 Feb, 2026 Reviewers invited by journal 19 Feb, 2026 Editor invited by journal 17 Feb, 2026 Editor assigned by journal 17 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 16 Feb, 2026 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. 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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-8723511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":595105304,"identity":"87fcc107-214f-4e76-875f-e419d0037286","order_by":0,"name":"Pradip Patil","email":"","orcid":"","institution":"Department of Chemical Sciences, Faculty of Science, Atmiya University, Rajkot","correspondingAuthor":false,"prefix":"","firstName":"Pradip","middleName":"","lastName":"Patil","suffix":""},{"id":595105305,"identity":"3822d0cb-3b7f-4aa8-af5d-8c88c83e056d","order_by":1,"name":"Bhavin Dhaduk","email":"","orcid":"","institution":"Department of Chemical Sciences, Faculty of Science, Atmiya University, Rajkot","correspondingAuthor":false,"prefix":"","firstName":"Bhavin","middleName":"","lastName":"Dhaduk","suffix":""},{"id":595105306,"identity":"996ac1b3-1822-47a3-aa60-e749745e733c","order_by":2,"name":"Pankajkumar Nariya","email":"","orcid":"","institution":"Department of Chemical Sciences, Faculty of Science, Atmiya University, Rajkot","correspondingAuthor":false,"prefix":"","firstName":"Pankajkumar","middleName":"","lastName":"Nariya","suffix":""},{"id":595105307,"identity":"04fb3d27-28dc-4145-8cb7-b4d514f798e2","order_by":3,"name":"Mahesh Savant","email":"data:image/png;base64,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","orcid":"","institution":"Department of Chemical Sciences, Faculty of Science, Atmiya University, Rajkot","correspondingAuthor":true,"prefix":"","firstName":"Mahesh","middleName":"","lastName":"Savant","suffix":""},{"id":595105308,"identity":"7a4aa728-6722-471d-81bc-235c96e9c3dd","order_by":4,"name":"Shamkumar P. Deshmukh","email":"","orcid":"","institution":"Department of Chemistry, D.B.F. Dayanand College of Arts and Science, Solapur, 413004","correspondingAuthor":false,"prefix":"","firstName":"Shamkumar","middleName":"P.","lastName":"Deshmukh","suffix":""}],"badges":[],"createdAt":"2026-01-28 16:41:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8723511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8723511/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103317680,"identity":"2d99bc14-df12-4205-8a02-50373ee7d258","added_by":"auto","created_at":"2026-02-24 11:12:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of Glyceryl Trinitrate (Nitroglycerin ) (NTG)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/ad00124961289db88577c1dc.png"},{"id":103317685,"identity":"4a0aece3-9d2a-4f84-9455-b42d5da0209a","added_by":"auto","created_at":"2026-02-24 11:12:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182256,"visible":true,"origin":"","legend":"\u003cp\u003eBlank Solution\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/7e7614fcc80b0c5294666736.jpeg"},{"id":103317659,"identity":"dfd99464-ab10-43fb-a600-f8542609e657","added_by":"auto","created_at":"2026-02-24 11:12:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120105,"visible":true,"origin":"","legend":"\u003cp\u003ePlacebo\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/d2ac94d021eb30dded3444ac.png"},{"id":103317679,"identity":"7a87a722-b565-4b1e-b3f0-e153cd7c461b","added_by":"auto","created_at":"2026-02-24 11:12:13","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222857,"visible":true,"origin":"","legend":"\u003cp\u003estandards Chromatogram\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/0c337aaad2bbdd9c009325ec.jpeg"},{"id":103317713,"identity":"101f9c6e-5497-44fd-bf25-44041f30b6f3","added_by":"auto","created_at":"2026-02-24 11:12:17","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":273711,"visible":true,"origin":"","legend":"\u003cp\u003eSpiked sample Chromatogram\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/21f1bf3d3425e3531231b24b.jpeg"},{"id":103317677,"identity":"640106ca-5b04-40e7-9577-170b63cfcc0c","added_by":"auto","created_at":"2026-02-24 11:12:12","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":209963,"visible":true,"origin":"","legend":"\u003cp\u003eControl sample Chromatogram\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/71f5d7bafe5fac3dbfb301c0.jpeg"},{"id":103506512,"identity":"25f244b8-3b82-49fc-ad04-2534fe09c370","added_by":"auto","created_at":"2026-02-26 13:37:14","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105254,"visible":true,"origin":"","legend":"\u003cp\u003eLinearity Graph\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/73bac47eac5cf8aadf9412a9.jpeg"},{"id":103511110,"identity":"130aa6f6-182a-458d-bd32-1bbbf5606f72","added_by":"auto","created_at":"2026-02-26 14:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2565050,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/447fb938-d555-407c-8203-8b27489b9f1b.pdf"},{"id":103317714,"identity":"f2b81653-b3dd-4172-9ce0-96efb4b6ed84","added_by":"auto","created_at":"2026-02-24 11:12:19","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":190061,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/ee323d9680f5cd408f25268e.jpg"},{"id":103317683,"identity":"9126857b-2563-49e6-baf6-f33a192ae8dd","added_by":"auto","created_at":"2026-02-24 11:12:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":95224,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8723511/v1/7b65b70d1b998142ba87ff64.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel Chromatographic Method Development and Validation for Nitroglycerin Determination in Semi-Solid Dosage Forms by RP-HPLC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitro-glycerine (NTG), also known as glyceryl trinitrate, is a potent organic nitrate compound extensively used in the treatment of cardiovascular conditions, primarily angina pectoris, heart failure and anorectal disorders such as chronic anal fissures [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. NTG functions as a potent vasodilator, exerting its therapeutic effect by releasing nitric oxide (NO), which relaxes vascular smooth muscle and reduces myocardial oxygen demand [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among the various dosage forms, topical ointments (4 mg/gm) offer a controlled and sustained release of the active pharmaceutical ingredient through transdermal absorption, minimizing first-pass metabolism and ensuring steady plasma levels over time [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to its volatile and chemically unstable, NTG might be degrades into metabolites such as 1,2-diniroglycerin and 1,3-diniroglycerin when exposed to the heat, light or moisture. This degradation compromises therapeutic efficacy and raises safety concerns [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it is crucial to develop a highly sensitive analytical method for the accurate quantification and characterization of NTG during manufacturing and product release to ensure the product's quality, safety, and efficacy throughout its shelf life.\u003c/p\u003e \u003cp\u003eA review of the literature reveals that various analytical techniques such as UV spectroscopy, HPLC, LC-MS, and GC-MS have been reported for the analysis of NTG and its mono and dinitrate metabolite across diverse matrices, including bulk drugs, pharmaceutical formulations, and human plasma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, most existing methods were time consuming, lack sensitive and unable to distinguish the decomposition products. Additionally, primarily focus on quantifying NTG without adequately addressing the regulatory requirements for controlling mono and dinitrate metabolites in Nitro-glycerine commercial dosage form [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Currently, no suitable analytical method is available for the simultaneous and sensitive detection of NTG and its mono and dinitrate metabolites in accordance with regulatory expectations. To address this gap, we have developed a highly selective HPLC method that significantly improves sensitivity compared to existing techniques. This method is particularly valuable for monitoring NTG and its dinitrate metabolite. All critical performance parameters including specificity, recovery, reproducibility, and linearity have been successfully validated. The method serves as a robust and reliable tool for routine quality control analysis, ensuring both the safety and regulatory compliance of NTG containing pharmaceutical products. The chemical structures of NTG and its dinitrate metabolite are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Method detail","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChemicals and Reagents\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll reagents and solvents used in this study were of analytical grade and were used without further purification. HPLC-grade methanol was procured from JT Baker, Mumbai, India. 30% hydrogen peroxide, sodium hydroxide and hydrochloric acid were purchased from Merck. Mumbai, India. Milli-Q water collected from the Milli-Q Plus water purification system (Millipore, Milford, MA, USA) was used throughout the analysis. An in-house working standard of NTG (potency 9.74%) and reference standards of its metabolites such as 1-mononitroglycerin (98.38%), 2-mononitroglycerin (95.44%), 1,2-dinitroglycerin (92.57%), and 1,3-dinitro glycerine (95.26%) were employed for method development, optimization, and validation. PTFE syringe (0.45 \u0026micro;m) filters were used for the filtration of the sample solutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of analytical solutions\u003c/strong\u003e\u003c/p\u003e\n\u003col style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003e\u003cstrong\u003ePreparation of standard stock solution of NTG (800 \u0026micro;g/mL):\u003c/strong\u003e Weight accurately 821.22 mg of NTG working standard to get effective 80 mg of pure NTG. Transfer the weighed material into 100 mL volumetric flask. Add about 70-80 mL of methanol and sonicate to dissolve. Dilute up to the mark with methanol and mix well.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePreparation of standard test solution of NTG (80 \u0026micro;g/mL):\u003c/strong\u003e Take 10 ml of standard stock solution of NTG into 100 ml volumetric flask and dilute up to the mark with methanol and mix well.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePreparation of sample solution of NTG pharmaceutical dosage form (40 \u0026micro;g/mL):\u0026nbsp;\u003c/strong\u003eAccurately weigh and transfer ointment equivalent to 4 mg of NTG into a 100 mL stoppered glass flask. Add approximately 50 mL of diluent using a calibrated pipette. Place the flask in a water bath maintained at \u003cstrong\u003e50 \u0026plusmn; 2 \u0026deg;C\u003c/strong\u003e for \u003cstrong\u003e12 min\u003c/strong\u003e, shaking the flask every 4 minutes to ensure uniform dispersion of the sample. After 12 min, shake the flask vigorously for 2 min to obtain a homogeneous viscous solution. Repeat the above process \u003cstrong\u003etwice\u003c/strong\u003e to ensure consistency. Transfer the final solution into a 20 mL glass syringe and filter through a \u003cstrong\u003e0.45 \u0026micro;m PVDF filter\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Discard the initial 4 mL of the filtrate to saturate the filter.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHomogeneity sample preparation:\u0026nbsp;\u003c/strong\u003eTo assess homogeneity, NTG ointment was sampled from multiple-dose containers. Samples were collected from the initial, middle, and end portions of each of five different marketed containers. The same sample preparation procedure described above was applied to each portion.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eInstrumentations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn HPLC system (Waters Alliance) comprising an integrated pump with inbuilt degasser, auto sampler, and column thermostat (Model: 2695 Separation Module), along with a variable UV wavelength detector (Model: 2489) and a photodiode array (PDA) detector (Model: 2998) from Waters Corporation (USA), was used for the detection of Nitro-glycerine (NTG). Data acquisition and processing (FR5) were carried out using Empower 3 software. The pH of the buffer solution was measured using a seven-excellence digital pH meter (Mettler Toledo, USA). Deaeration of the mobile phase was performed using a USB 6.5 L ultrasonic bath (PCi Analyst).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatographic condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromatographic separation was carried out using an -C18 column (150 mm \u0026times; 4.6 mm, 5 \u0026micro;m particle size) L1- Octadecylsilane chemically bonded to porous or non-porous silica or ceramic microparticles, 1.5 to 10\u0026micro;m in diameter . The mobile phase consisted of purified water and methanol in a 60:40 (v/v) ratio, delivered in isocratic mode at a flow rate of 1.0 mL/min. The column was maintained at ambient temperature, and the injection volume was set to 20 \u0026micro;L. The total run time for each analysis was 15.0 min. The optimized chromatographic parameters used for the effective separation of NTG and its metabolites are summarized in \u003cstrong\u003eTable 1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable :1 chromatographic parameter\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"627\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCondition\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eMode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eHPLC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eColumn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eC18 4.6 X 150 mm , 5 \u0026micro;m (L1 packing )\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eWavelength\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e220 nm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eFlow rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e1.0 ml/ min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eInjection volume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e20 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eColumn over temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eAmbient\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eRun time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e15 min the retention of Glyceryl Trinitite Peak\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 627px;\"\u003e\n \u003cp\u003esystem suitability :\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eUSP Tailing factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eNot more than 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eUSP Plate count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eNot less than 2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003e% RSD of Five standards injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 313px;\"\u003e\n \u003cp\u003eNot more than 2.0 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eForce degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForced degradation studies were performed on Nitro-glycerine (NTG) ointment at a concentration equivalent to 4 mg/gram to evaluate the stability and specificity of the developed HPLC method. Samples were subjected to various stress conditions, including acid hydrolysis, base hydrolysis, oxidation, thermal, humidity, and photolytic degradation. Placebo and blank were also evaluated to confirm no interference at the retention time of NTG and known impurities. After the designated exposure periods, samples were neutralized as necessary, diluted, and analyzed using the developed HPLC method with PDA detection. Peak purity was assessed using 3D-PDA data, where the \u003cstrong\u003epurity angle\u003c/strong\u003e was compared against the \u003cstrong\u003epurity threshold\u003c/strong\u003e to evaluate the homogeneity of the NTG peak.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMethod development and optimization\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore initiating the development of the LC method for the quantification of NTG in topical ointment formulations, essential information about the compound was gathered from literature and experimental data. NTG is a clear, colourless to slightly yellow liquid with a melting point of approximately 14 \u0026deg;C and a boiling point near to 50 \u0026deg;C. It is miscible with acetone and anhydrous ethanol, and possesses a pKa of 5.6, while exhibiting a neutral pH of 7. 0 [17]. Due to the polar nature, reverse phase chromatography was chosen for method development.\u003c/p\u003e\n\u003cp\u003eThe primary objective was to develop a selective and robust LC method that could effectively separate and quantify NTG from formulation excipients. A major challenge was to achieve appropriate retention with symmetrical peak shape and absence of placebo interference. Considering these requirements, multiple chromatographic trials were carried out by varying mobile phase composition, column type, and system parameters to determine suitable separation conditions. The wavelength of 220 nm was selected for detection based on UV spectral scan of a standard solution (80 \u0026micro;g/mL), which demonstrated optimal absorbance and peak purity.\u003c/p\u003e\n\u003cp\u003eIn the initial trial, a mobile phase consisting of methanol:water (50:50, v/v) was tested on an Inertsil C18 column. A distinct peak was obtained at 10.5 min, failed to comply with USP \u0026lt; 621 \u0026gt; system suitability criteria. In the second trial, a Hypersil BDS C18 column was used with a modified mobile phase ratio of methanol:water (60:40, v/v) and a flow rate of 1.3 mL/min. The result showed a distinct peak of NTG with retention time of 7.7 min; however, system suitability parameters were not met. A third trial using an Alpha C18 column (4.6 \u0026times; 150 mm, 5 \u0026micro;m) under similar conditions yielded a retention time of 5.5 min, but failed to comply with USP \u0026lt; 621 \u0026gt; system suitability criteria. To overcome these limitations, further optimization was performed using an C18 column (4.6 \u0026times; 150 mm, 5 \u0026micro;m) L1 Packing with methanol:water (60:40, v/v) as the mobile phase, a flow rate of 1.0 mL/min, injection volume of 50 \u0026micro;L, and a column temperature maintained at 25 \u0026deg;C . The resulting chromatogram showed a well-resolved NTG peak at 5.5 minutes with excellent peak symmetry, and acceptable theoretical plate count and tailing factor. This optimized chromatographic condition was selected as the final method for further validation as per ICH guidelines. The finalized method demonstrated robustness, reproducibility, and compliance with all system suitability criteria, ensuring its suitability for routine quantification of NTG in pharmaceutical formulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proposed method was validated for specificity, precision, accuracy, linearity, and limits of detection and quantification according ICH Q2 (R2) guidelines [19-22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSystem suitability testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystem suitability testing was conducted to confirm that the analytical system was fit for its intended purpose at the time of analysis. This evaluation involved six replicate injections (n = 6) of a NTG standard test solution at a concentration of 80 \u0026micro;g/ml. The average retention time for NTG was 5.71 min, with a % RSD of peak areas calculated at 0.43%. Since the % RSD was well within acceptable limits, the system was deemed suitable for analysis [23, 24]. A summary of these results is presented in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eResults of validation parameters: a) System suitability b) Specificity c) Linearity d) Recovery / accuracy e) Method Precision (Inter / Intra)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"661\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eParameters\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcceptable criteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 661px;\"\u003e\n \u003cp\u003e\u003cem\u003eSystem suitability\u0026nbsp;\u003c/em\u003e\u003cem\u003e(Mean \u0026plusmn; RSD) (n =6)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Retention time\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e%RSD \u0026le; 2.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.41 \u0026plusmn; 0.43 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;USP tailing factor\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026le; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.2 \u0026plusmn; 3.15 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;USP plate count\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026ge; 2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7329 \u0026plusmn; 0.67 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Standard area\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e%RSD \u0026le; 2.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e17702 \u0026plusmn; 0.67 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSpecificity\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo interference at retention time of NTG or known impurities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo interference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 661px;\"\u003e\n \u003cp\u003e\u003cem\u003eLinearity (40 - 120\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026micro;g/ml)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Regression equation\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLine equation should show good fit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3E-08x + 0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Slope\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePositive slope indicating sensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3E-08x\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026ge; 0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.9993\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 661px;\"\u003e\n \u003cp\u003e\u003cem\u003e% Recovery (Mean \u0026plusmn; RSD)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Level-1 (50%)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e98.0% \u0026ndash; 102.0%, RSD \u0026le; 2.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100.1 \u0026plusmn; 0.20 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Level-2 (100%)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.3 \u0026plusmn; 0.32 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Level-3 (150%)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.9 \u0026plusmn; 0.07 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePrecision (Mean \u0026plusmn; RSD)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Intraday (n = 6)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e%RSD \u0026le; 2.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1902837.17 \u0026plusmn; 0.87 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Inter day\u0026nbsp;\u003c/em\u003e\u003cem\u003e(n = 6)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1911050.67 \u0026plusmn; 0.35 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: R\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= determination of coefficients, RSD = Relative standard deviation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specificity of the method was evaluated by assessing potential interfering components in the HPLC spectrogram. A 80 \u0026micro;L solution of blank, standard test solutions of 1-Mononitroglycerin, 2-Mononitroglycerin, 1,2-Dinitroglycerin, and 1,3-Dinitroglycerin and NTG, and a spiked sample (containing both NTG and its metabolites) were injected separately into the HPLC system. The results demonstrated no interference from any interfering components, and all analyte peaks remained distinct and unaltered. The spectrogram obtained are shown in Figure 2 to 6. The results revealed that there was no interference of the NTG with its metabolites peaks and hence the specificity of the developed method was proven.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2 to 5 \u0026ndash; HPLC Assay Chromatogram :\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe linearity of the NTG was satisfactorily demonstrated with a five-point calibration graph, ranging from the 50 to 150 % of the specification level (actual concentration: 0.04, 0.06, 0.08, 0.10, and 0.12, \u0026mu;g/mL). The slope, intercept and correlation coefficient values were derived from linear least-square regression analysis and the data is presented in Table 2 and figure 6. It reveals that an excellent correlation existed between the peak area concentrations of NTG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recovery testing involved spiking a known quantity of NTG with placebo at various levels (50% = 40 \u0026mu;g/mL, 100% = 80 \u0026mu;g/mL, and 150% = 120 \u0026mu;g/mL of the specification). Each concentration level was injected triplicate into the HPLC. The % recovery values obtained for the NTG ranged from 99.3% to 100.1%, which are within the acceptance criteria. The % RSD values of recoveries for the NTG ranged from 0.07 % to 0.32 % at 50 to 150 % levels. The % recovery results are shown in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA precision study was conducted by injecting a standard test samples (n = 6) of NTG to evaluate intra and inter day precision. The % RSD obtained for intra, and inter day precision were 0.87, and 0.35 %, respectively. These results demonstrate that the method is sufficiently precise, with detailed precision data provided in Table 2 and Table 3 a and 3b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable No 3a; Results of Inter and Intra days precision\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"98%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterday\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eInjection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1916781\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1896798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1930011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1889865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1892314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1891254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1902837.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.873\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eIntraday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eMorning\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003eEvening\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e%RSD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eInjection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1910023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1916358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1910012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1912817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1901293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1921201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e1911050.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.352\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eB ) Method precision and Homogeneity : Method precision was verified by analysing the six samples of the assay with bath analysis and Initial , middle and bottom in triplicate samples as per method and results are tabulated in Table 7 and 8 .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable No 3b: Results of Method precision and Homogeneity\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSample No\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;% Assay Nitroglycerin (Glyceryl Trinitite)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Assay Nitroglycerin (Glyceryl Trinitite for homogeneity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e99.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eInitial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e99.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eMiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e98.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;Mean\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eEnd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e97.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e% RSD\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eLimit\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 189px;\"\u003e\n \u003col\u003e\n \u003cli\u003e% Assay Nitroglycerin (Glyceryl Trinitite) -90.0 to 105.0 %\u003c/li\u003e\n \u003cli\u003e% RSD of 6 samples not More than 2.0\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e98.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e% RSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eLimit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003col\u003e\n \u003cli\u003e% Assay Nitroglycerin (Glyceryl Trinitite) -90.0 to 105.0 %\u003c/li\u003e\n \u003cli\u003e% RSD of 9 samples not More than 2.0\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSample homogeneity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample homogeneity of NTG ointment (4 mg/g) was confirmed by analyzing six assay samples taken from the beginning, middle, and end of a batch. The mean assay was 99.1% with a %RSD of 0.73%, indicating uniform API content throughout. Method precision and homogeneity data are shown in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4:\u0026nbsp;\u003c/strong\u003eMethod precision and sample homogeneity data of NTG ointment (4 mg/g)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSample No.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Assay \u0026ndash; Method Precision\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSampling Location\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Assay \u0026ndash; Sample Homogeneity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eInitial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eMiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eEnd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e97.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMean\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e% RSD\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.73\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcceptance Criteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e90.0% \u0026ndash; 105.0%,\u003c/p\u003e\n \u003cp\u003e% RSD \u0026le; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e90.0% \u0026ndash; 105.0%, %\u003c/p\u003e\n \u003cp\u003eRSD \u0026le; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe robustness of the HPLC method for Nitroglycerin (NTG) assay was assessed by introducing deliberate variations in flow rate, column temperature, detection wavelength, sample heating time and temperature, and mobile phase composition. As summarized in Table 5, all assay results remained within the acceptable range of 98.0 - 102.0 %, with %RSD values below 2.0%. System suitability parameters including USP tailing (\u0026le; 2.0) and plate count ( \u0026ge; 2500) were consistently met. Although a slight variation was noted under altered heating temperature conditions (absolute difference = 2.0 %), it remained within acceptable limits, confirming the method\u0026apos;s robustness [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003eResult of Robustness study\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSr. No\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChange in parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUSP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTailing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUSP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePlate count\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Recovery Assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% RSD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% difference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow rate (ml/min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColumn temperature (\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e6912\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWavelength (nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample heating time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7376\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e99.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample heating temperature (\u0026deg;C)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e96.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e55.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNobile phase composition - Methanol : Water (% v/v)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e50:50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e6895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e60:40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e70:30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e6843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eForce degradation study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNTG ointment samples were subjected to various stress conditions, including acid hydrolysis, base hydrolysis, oxidation, thermal, humidity, and photolytic degradation [26]. It was observed that NTG was stable under all tested stress conditions including acid, oxidative, thermal, photolytic, and humidity degradation, with minor degradation (\u0026lt;5%). Base hydrolysis resulted in moderate degradation (3.4 %), but peak purity remained acceptable, confirming the method\u0026apos;s specificity and stability-indicating capability. No interfering peaks were observed in the blank or placebo chromatograms at the NTG retention time. The force degradation results are summarized in Table 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6:\u0026nbsp;\u003c/strong\u003eForce degradation results for NTG Ointment (4 mg/g)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSr. No\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode of Degradation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCondition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Assay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsolute Degradation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePurity Angle\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePurity Threshold\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePurity Flag\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eAcid Hydrolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e2 mL of 0.25 N HCl at 25 \u0026deg;C / 20 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e95.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eBase Hydrolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e2 mL of 0.01 N NaOH at 50 \u0026deg;C / 20 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eOxidative Degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e2 mL of 30% H₂O₂, 25 \u0026deg;C / 24 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e97.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.345\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eHumidity Degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e25 \u0026deg;C / 90% RH for 7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003ePhotolytic Degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e1.2 Million Lux Hours, 200 W\u0026middot;hr/m\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eThermal Degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e40 \u0026deg;C for 48 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e98.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eApplication of the method for real sample analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree commercial-scale batches of NTG topical ointment (4 mg/g) were analyzed using the developed RP-HPLC method. Test sample solutions were prepared according to the validated procedure and subjected to chromatographic analysis. The results confirmed the presence of NTG at the labelled concentration in all three batches, with no detectable levels of mono or dinitrate impurities. To assess method specificity, known amounts of NTG mono- and dinitrate impurities were spiked into the test samples. The method successfully separated all analytes without interference from the formulation matrix. The spiked impurities were recovered within acceptable limits (98.5%\u0026ndash;101.2%), confirming that method is suitable for routine quality control of bulk and finished products. The results are summarized in Table 2 and 3a and 3b .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of the present method with the earlier reported methods\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analytical performance of the proposed RP-HPLC method was evaluated and compared with previously reported techniques in terms of sensitivity, accuracy, specificity and precision run time, and selectivity (Table 2). Unlike earlier methods, which focused developed on quantification of NTG in different matrices, the present method is specifically developed for the identification and quantification of a NTG and its mono and dinitrate metabolites in 4 mg/g Topical Ointment, in accordance with ICH Q2(R2) guidelines. Moreover, none of the existing reported methods address this specific impurity. The proposed method offers shorter run time (15 min) and uses low environmental impact reagents, and demonstrates improved selectivity, high accuracy, and cost-effectiveness, making it valuable advancement for impurity control in pharmaceutical.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Greenness of Analytical HPLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe greenness of the developed RP-HPLC method for the quantification of NTG and its nitrate metabolites was evaluated using three complementary metrics: \u003cstrong\u003eAnalytical Eco-Scale\u003c/strong\u003e\u003cstrong\u003e, \u003cstrong\u003eAGREE\u003c/strong\u003e,\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eGAPI\u0026nbsp;\u003c/strong\u003e\u003c/strong\u003e\u003cstrong\u003e[27]\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eEco-Scale score of 87\u0026nbsp;\u003c/strong\u003e\u003c/strong\u003eTable S1 classifies the method as excellent in terms of greenness. The \u003cstrong\u003eAGREE tool yielded a score of 0.63\u003c/strong\u003e, indicating good alignment with green analytical chemistry principles, particularly due to its low energy usage and direct, multi-analyte analysis. The \u003cstrong\u003eGAPI pictogram\u003c/strong\u003e revealed a balanced profile, with strengths in instrumentation and direct measurement but concerns due to solvent consumption and heating during sample preparation. Overall, the method qualifies as environmentally acceptable and suitable for routine analytical applications, with scope for further improvement by adopting greener solvents. The greenness scores for the proposed method were evaluated and presented in Table S2.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA robust, precise, and stability-indicating RP-HPLC method was successfully developed and validated for the quantification of NTG in 4 mg/g ointment formulations. The method employed a simple isocratic elution with water and methanol (60:40, v/v) on a C18 column, offering good resolution and a retention time of approximately 5.7 minutes. The method demonstrated excellent linearity, precision, and accuracy across the specified concentration range (40\u0026ndash;120 \u0026micro;g/mL), with recovery rates between 99.3% and 100.1%. Forced degradation studies confirmed the method's specificity and its ability to distinguish NTG from its degradation products and metabolites, without interference. The peak purity analysis via PDA confirmed the homogeneity of the NTG peak under stress conditions. Additional validation parameters such as filter compatibility, solution and mobile phase stability, homogeneity, and robustness further confirmed the reliability and suitability of the method for routine quality control analysis. Overall, this validated method meets all the ICH Q2(R2) criteria and is well-suited for the routine release testing and stability studies of NTG ointment, ensuring compliance with regulatory expectations and safeguarding product quality and efficacy throughout its shelf life.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author gratefully acknowledges \u003cstrong\u003eDepartment of Chemical Sciences, Faculty of Science, Atmiya University\u0026nbsp;\u003c/strong\u003efor their generous support, valuable guidance, and for providing all the necessary facilities that contributed significantly to the successful completion of this work and Jegan Balan for analytical resources and execution help.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePradip Patil:\u0026nbsp;Project administration, Methodology, Investigation, conducting the experiments,\u0026nbsp;Bhavin Dhaduk:\u0026nbsp;Analysis data acquisition, Writing.\u0026nbsp;Pankajkumar Nariya and Shamkumar Deshmukh:\u0026nbsp;Investigation, Methodology, Funding acquisitions.\u0026nbsp;Mahesh Savant:\u0026nbsp;Writing review and editing data acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was self-funded by the authors. No external financial support was received for the conduct of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are included within this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or animals. Therefore, ethical approval was not required. This study did not involve human participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not contain any individual person\u0026rsquo;s data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTwiner MJ, Hennessy J, Wein R, Levy PD (2022) Nitroglycerin use in the emergency department: current perspectives. Open Access Emerg Med \u003cstrong\u003e14\u003c/strong\u003e:327\u0026ndash;333.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYousif EM et al (2012) Formulation and clinical testing of glyceryl trinitrate ointment for treatment of anal fissures. Am J Pharmacol Toxicol.\u003c/li\u003e\n \u003cli\u003eLund JN, Scholefield JH (1997) A randomised, prospective, double-blind, placebo-controlled trial of glyceryl trinitrate ointment in treatment of anal fissure. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e349\u003c/strong\u003e:11\u0026ndash;14.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDivakaran S, Loscalzo J (2017) The role of nitroglycerin and other nitrogen oxides in cardiovascular therapeutics. J Am Coll Cardiol.\u003c/li\u003e\n \u003cli\u003eBenson HAE et al (2019) Topical and transdermal drug delivery: from simple potions to smart technologies. Curr Drug Deliv.\u003c/li\u003e\n \u003cli\u003eFriciu M et al (2016) Stability of hydrocortisone, nifedipine, and nitroglycerine compounded preparations for the treatment of anorectal conditions. Can J Hosp Pharm.\u003c/li\u003e\n \u003cli\u003eScheife AH et al (1982) Stability of intravenous nitroglycerin solutions. J Pharm Sci.\u003c/li\u003e\n \u003cli\u003eSaadallah MS et al (2025) Stability and characteristics of nitroglycerin sublingual tablets available in the Iraqi market. J Pharm Innov.\u003c/li\u003e\n \u003cli\u003eMartel R et al (2010) Determination of nitroglycerin and its degradation products by solid-phase extraction and LC\u0026ndash;UV. Chromatographia.\u003c/li\u003e\n \u003cli\u003ePo ALW, Irwin WJ (1980) High-performance liquid chromatography techniques and applications. J Clin Pharm Ther.\u003c/li\u003e\n \u003cli\u003eGelber L (1980) High-performance liquid chromatographic analysis of nitroglycerin ointment. J Pharm Sci.\u003c/li\u003e\n \u003cli\u003eOlsen CS, Scroggins HS (1983) High-performance liquid chromatographic determination of nitroglycerin in sublingual, sustained-release, and ointment dosage forms. 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Profiles Drug Subst Excip Relat Methodol.\u003c/li\u003e\n \u003cli\u003eKorikana SP et al (2026) Development and validation of a stability-indicating LC method for the determination of empagliflozin-related substances with sensitivity profiling and robustness assessment using analytical quality by design principles. Sep Sci Plus.\u003c/li\u003e\n \u003cli\u003eFauzee AFB, Walker RB (2013) Forced degradation studies of clobetasol 17-propionate in methanol, propylene glycol, as bulk drug and cream formulations by RP-HPLC. J Sep Sci.\u003c/li\u003e\n \u003cli\u003eShi M et al (2023) Overview of sixteen green analytical chemistry metrics for evaluation of the greenness of analytical methods. TrAC Trends Anal Chem.\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-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nitroglycerin, RP-HPLC, Stress studies, Validation, Routine analysis","lastPublishedDoi":"10.21203/rs.3.rs-8723511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8723511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe method was validated according to ICH Q2(R2) guidelines included the system suitability, specificity, linearity, accuracy, homogeneity, robustness and forced degradation. The report exhibits a robust and stability-indicating RP-HPLC method developed for the quantification of Nitroglycerin in 4 mg/g ointment formulations. Chromatographic separation was performed using an L1 packing C18 column (150 mm \u0026times; 4.6 mm, 5 \u0026micro;m) with an isocratic mobile phase of water and methanol in a 60:40 (v/v) ratio. The flow rate was maintained at 1.0 mL/min, and detection was carried out using a UV detector set at 220 nm and run time was 15.0 min. It revealed excellent system suitability, specificity without interference from excipients, impurities, degradants, or matrix components and linearity over the range of 40\u0026ndash;120 \u0026micro;g/mL. Percentage accuracy (recovery) values obtained for the NTG is from 99.3% to 100.1%. Photodiode array (3D-PDA) detector unveils homogeneity of the NTG peak under stress conditions confirmed uniform distribution across formulation layers. Robustness process confirmed method reliability, method transfer and reduced errors and reliability under varied conditions. Forced degradation study reveal specificity and its ability to distinguish NTG from its degradation products and metabolites, without interference under the various stress conditions such as acid and base hydrolysis, oxidation, thermal, humidity, and photolytic degradation. The assessment of greenness of analytical HPLC was carried out using Analytical Eco-Scale, AGREE, and GAPI. The Eco-Scale score of 87, the AGREE tool yielded a score of 0.63 and the GAPI pictogram revealed a balanced profile. Therefore, such worthwhile quantification study intended for regulatory compliance approach in the pharmaceutical and healthcare avenue.\u003c/p\u003e","manuscriptTitle":"Novel Chromatographic Method Development and Validation for Nitroglycerin Determination in Semi-Solid Dosage Forms by RP-HPLC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 11:11:09","doi":"10.21203/rs.3.rs-8723511/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-09T09:34:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T09:20:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T01:08:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210879209622587702657258771157741622314","date":"2026-02-27T16:10:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269509742945613960155438369798990528573","date":"2026-02-25T08:10:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T06:53:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324197851638640361451182164232907456751","date":"2026-02-22T11:02:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-19T07:55:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-17T15:03:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T11:18:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T05:35:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2026-02-16T05:31:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea53fd33-ccc1-410d-b254-1b6367e27dea","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T09:25:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 11:11:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8723511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8723511","identity":"rs-8723511","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}