Design and validation of a robust stability-indicating reversed-phase HPLC method for quantification of mesalamine in formulated drug products

Abstract

A reliable and sensitive RP-HPLC method was developed and validated for the accurate quantification of mesalamine in bulk and formulated pharmaceutical products. The analysis was carried out on a C18 column (150 mm × 4.6 mm, 5 μm) using a mobile phase of methanol: water (60:40 v/v), with a flow rate of 0.8 mL/min, and UV detection at 230 nm. Methanol: water (50:50 v/v) was used as the diluent. The method demonstrated excellent linearity across the concentration range of 10–50 µg/mL (y = 173.53x – 2435.64, R² = 0.9992), high accuracy with recoveries between 99.05% and 99.25% (%RSD < 0.32%), and outstanding precision with intra- and inter-day %RSD values below 1%. Robustness was confirmed under slight method variations (%RSD < 2%), and LOD and LOQ were found to be 0.22 µg/mL and 0.68 µg/mL, respectively. Forced degradation studies under acidic, basic, oxidative, thermal, and photolytic stress confirmed the method’s specificity and stability-indicating capability. Assay of a commercial mesalamine tablet (Mesacol^®, 800 mg label claim) showed a recovery of 99.91%, validating the method’s applicability for routine quality control and regulatory compliance.

Graphical Abstract

Introduction

Mesalamine, alternatively referred to as mesalazine or 5-aminosalicylic acid (5-ASA), is a well-established, bowel-specific anti-inflammatory agent widely used in the treatment of inflammatory bowel diseases (IBD), particularly ulcerative colitis and mild-to-moderate forms of Crohn’s disease. Owing to its therapeutic efficacy in attenuating intestinal inflammation, mesalamine remains central to the management of disease-related symptoms, including abdominal pain, diarrhea, and rectal bleeding, while concurrently promoting mucosal healing and improving long-term clinical outcomes [1]. Given its narrow therapeutic window, chemical sensitivity, and chronic usage profile, the accurate quantification and stability monitoring of mesalamine in pharmaceutical formulations is essential to ensure consistent clinical efficacy and regulatory compliance. Pharmacologically, mesalamine is an amino salicylate derivative that exerts its therapeutic action locally within the gastrointestinal tract. This site-specific mechanism of action results in minimal systemic absorption, thereby substantially reducing the likelihood of systemic adverse effects a critical consideration in long-term IBD management. In addition to its anti-inflammatory function, mesalamine exhibits intrinsic antioxidant capacity, acting as a free radical scavenger that mitigates oxidative stress-induced mucosal damage, a key pathogenic factor in IBD progression [2]. This dual mechanism—comprising anti-inflammatory and antioxidant pathways forms the basis of its therapeutic efficacy in both induction and maintenance of remission, as well as in the prevention of disease flare-ups.Mesalamine also constitutes the active moiety of sulfasalazine, a prodrug metabolized in the colon by bacterial azoreductases to release mesalamine and sulfapyridine. While mesalamine confers therapeutic benefits, sulfapyridine has been associated with gastrointestinal and systemic adverse effects. This pharmacokinetic distinction has led to the preference for mesalamine-only formulations, particularly in patients who experience sulfasalazine intolerance. Consequently, accurate and robust analytical methods are critical for the quantification of mesalamine across various pharmaceutical matrices, both during manufacturing and post-market surveillance [3]. To meet this need, various analytical techniques have been developed for the determination of mesalamine in bulk drug substances, pharmaceutical formulations, and biological fluids. These methods range from conventional approaches such as UV–Visible spectrophotometry to more advanced technologies like high-performance liquid chromatography (HPLC). Among them, reverse-phase HPLC (RP-HPLC) has become the preferred method due to mesalamine’s moderate polarity, favorable UV absorbance properties, and excellent compatibility with aqueous-organic mobile phases, enabling precise, accurate, and reproducible detection [4].

Pharmaceutical stability, as defined by the International Council for Harmonisation (ICH), refers to the ability of a drug product to retain its physical, chemical, microbiological, therapeutic, and toxicological integrity throughout its designated shelf life under specified storage conditions. Stability is a critical quality attribute that directly impacts drug efficacy, patient safety, and product lifecycle management [5]. It encompasses the duration from the date of manufacture during which the drug maintains acceptable potency and does not undergo significant alterations in appearance, performance, or safety. In accordance with ICH guideline Q1A(R2), forced degradation studies—also referred to as stress testing are essential components of stability assessment and method validation. These studies expose the active pharmaceutical ingredient (API) to extreme environmental conditions, including elevated temperature, humidity, oxidative stress, photolysis, and hydrolysis across a wide pH spectrum. Such conditions simulate long-term storage or accelerated degradation scenarios, thereby revealing potential degradation pathways and intrinsic chemical liabilities. A stability-indicating analytical method (SIAM) must therefore be capable of accurately quantifying the intact drug while concurrently resolving, detecting, and identifying its degradation products. These capabilities are essential to ensure robust quality control and regulatory acceptance throughout drug development, manufacturing, and storage [6]. The design and validation of highly sensitive, precise, and stability-indicating analytical methods for mesalamine not only fulfill regulatory requirements but also support the delivery of safe, effective, and high-quality treatments in clinical practice. These analytical strategies are fundamental to ensuring therapeutic consistency in the long-term management of chronic gastrointestinal disorders such as IBD.

Materials and methodology

HPLC instrument

The quantitative analysis was carried out using a Shimadzu UFLC system equipped with an LC-20AD binary pump and SPD-20 A UV-Visible detector. A manual injector system was employed for sample introduction. The LC-20AD pump, designed with a dual-plunger, parallel-type configuration, enabled highly precise solvent delivery within a flow rate range of 0.0001 to 10.000 mL/min, maintaining accuracy within ± 1% and repeatability (%RSD) below 0.06%. This setup ensured excellent reproducibility and consistent chromatographic performance throughout the analysis. A reverse-phase C18 column (ODS, 150 mm × 4.6 mm, 5 μm) was employed. The mobile phase consisted of methanol and water in the ratio of 60:40 (v/v), degassed by ultrasonication for 5 min before use. The flow rate was maintained at 0.8 mL/min, and the injection volume was 20 µL. Detection was performed at 230 nm using a UV-Visible detector. The run time was 10 min, and the diluent used for all preparations was methanol: water (50:50 v/v).

Chemicals and reagents

Mesalamine (API, purity 99.8%) was generously supplied by Aurobindo Pharma Ltd., Hyderabad, India. HPLC-grade methanol, acetonitrile, and water were procured from Merck Life Science Pvt. Ltd., Bengaluru, India. A 3% hydrogen peroxide solution (IP grade) was obtained from SD Fine-Chem Limited, Mumbai, India. Mesacol^® tablet as “800 mg of mesalamine per tablet” were sourced from Wellness Forever Pharmacy, Pune.

Preparation of calibration standards

A 10 mg quantity of mesalamine reference standard was accurately weighed, dissolved in diluent with sonication, and diluted to 10 mL to obtain a 1 milligram per milliliter stock solution. From this, an aliquot of 0.1 mL was further diluted to prepare a 10 µg/mL working solution and subsequently filtered through a 0.45 μm membrane filter before analysis. A seven-point calibration curve was established using serial dilutions to achieve concentrations of 20, 25, 30, 35, and 50 µg/mL, with each level injected in triplicate. Calibration solutions were freshly prepared daily and immediately analyzed to ensure precision and prevent degradation, following standard protocols reported by Moharana et al. [7], who developed and validated an RP-HPLC method for mesalamine using similar concentration ranges and preparation techniques.

Evaluation of mesalamine stability under stress conditions

Stress degradation studies were performed to assess the stability of mesalamine under different stress environments. Acid-induced degradation was assessed by incubating a solution of mesalamine with 0.1 N HCl at 25 ± 2 °C for 2 h, followed by neutralization with 0.1 N NaOH, dilution, filtration, and RP-HPLC analysis. Similarly, alkaline degradation was evaluated by treating the solution with 0.1 N NaOH, neutralizing with 0.1 N HCl after 2 h, and analyzing via RP-HPLC. Oxidative degradation was studied by exposing the solution to 3% hydrogen peroxide under similar conditions before filtration and analysis. Thermal degradation involved subjecting pure mesalamine to 80 °C dry heat for 24 h, reconstituting it with diluent, and analyzing by RP-HPLC. Photolytic stability was evaluated by subjecting the solid drug to ultraviolet (UV) exposure at 254 nm for 24 h according to ICH Q1B guidelines, followed by dissolution, filtration, and analysis. In all cases, solutions were membrane filtered (0.45 μm) prior to chromatographic evaluation using the validated RP-HPLC method to determine the extent of degradation and verify the method’s specificity, consistent with procedures described by Walash et al. [8] and in accordance with ICH guidelines [9].

Method validation

Analytical linearity and standard curve generation

All method validation studies were performed in accordance with ICH Q2(R2) guidelines. This includes assessments of linearity, accuracy, precision, robustness, LOD, LOQ, and specificity. Linearity denotes the ability of an analytical method to produce results that are directly proportional to the concentration of the analyte within a given range, as defined by the ICH Q2(R1) guidelines [10]. This characteristic ensures that the method can accurately quantify the analyte across a range of concentrations typically encountered in routine analysis. Linearity may be established either by performing serial dilutions of a stock solution or by analyzing independently prepared concentrations of the analyte using the proposed analytical method [11]. In the present study, the linearity of mesalamine was assessed over a predefined concentration range. A calibration curve was constructed by plotting the mean peak area (from triplicate injections) against the corresponding concentrations of mesalamine. The linear relationship was evaluated using linear regression analysis based on the least squares method. The resulting calibration parameters including the slope, y-intercept, and coefficient of determination (r²) were determined, demonstrating a strong linear correlation between analyte concentration and detector response, which confirms the method’s suitability for quantitative analysis.

Accuracy

The accuracy of the method was determined according to the ICH Q2(R1) guidelines using the standard addition procedure [10]. Previously examined mesalamine API samples were spiked with known amounts of the standard solution at three concentration levels typically representing 80%, 100%, and 120% of the target analyte concentration. These fortified samples were then analyzed using the validated chromatographic method. Accuracy was evaluated by calculating the percentage recovery of the added analyte at each concentration level, along with the %RSD to determine the precision of the recovery values. The recovery percentages across all levels were within the acceptable range of 98–102%, and the %RSD values were well below 2%, meeting the acceptance criteria outlined in validation protocols. These results demonstrate that the method is accurate, reliable, and well-suited for the quantitative analysis of mesalamine in both bulk drug substances and finished pharmaceutical formulations. Similar accuracy performance using the standard addition method for mesalamine was reported by Moharana et al. [12], further supporting the validity of the approach. The current study demonstrates improved sensitivity, better linearity (R² = 0.9992), and validated robustness and specificity. Additionally, our method includes comprehensive forced degradation assessments aligned with ICH Q2(R2) guidelines, which were not consistently covered in prior studies. This comparative evaluation has been added to the discussion section to highlight the advancements of the present method over existing literature.

Precision

The degree of precision of the analytical method was assessed in accordance with the ICH Q2(R1) guidelines [10] and evaluated at two levels: intra-day (repeatability) and inter-day (intermediate precision). Repeatability was assessed by analyzing three concentrations of mesalamine API each within the validated linear range in triplicate on the same day under consistent experimental conditions. Intermediate precision was evaluated by repeating the same procedure over three consecutive days, using the same instruments, reagents, and analyst. This two-tiered approach ensures that the method yields reproducible results across both short-term and day-to-day variations, in line with the validation methodology described by Taverniers et al. [11], who emphasized the importance of assessing both precision tiers for reliable analytical performance in pharmaceutical assays.

Robustness

The resilience of the analytical approach was evaluated to ascertain its robustness against minor, intentional variations in analytical conditions, as stipulated by ICH Q2(R1) guidelines. This evaluation guarantees the method’s dependability during standard operations under slightly modified conditions. The robustness of the method was assessed by deliberately varying flow rate (0.7 and 0.9 mL/min), column temperature (± 2 °C), and wavelength (± 2 nm). System suitability parameters including retention time, tailing factor, and theoretical plates were monitored. All variations showed %RSD below 2%. The method consistently produced satisfactory results across all tested conditions, demonstrating that its performance was not significantly affected by these modifications. These findings confirm the method’s robustness, consistency, and suitability for routine quality control testing [13].

Assessment of detection limit (LOD)

The LOD signifies the minimum amount of a sample that can be consistently distinguished from background noise, but exact measurement may not be achievable under specified experimental conditions. In chromatographic evaluations, the LOD was estimated employing a statistically validated procedure that employed the standard deviation of the y-intercepts from the regression analysis of the standard curve and the slope of that curve. The Level of Detection (LOD) was determined using the following equation: . This formula provides a robust estimation of the method’s sensitivity, ensuring that even trace levels of the analyte can be confidently detected, thus supporting the analytical reliability of the method employed [14].

Assessment of quantitation limit (LOQ)

The LOQ represents the minimum quantity of a sample that can be measured with correctness and consistency under defined experimental circumstances. This study established the LOQ utilizing the following equation:

   where Sa represents the standard deviation of the response (typically the peak area) from replicate injections, and b represents the slope of the calibration curve. This statistically sound approach ensures the method’s reliability for precise quantification, particularly at lower analyte concentration levels, thereby reinforcing its suitability for routine analytical applications [14].

Specificity

The analytical method’s specificity was assessed by exposing mesalamine to forced degradation under diverse stress conditions to ensure it could accurately distinguish the drug from its potential degradation products. These conditions included acidic hydrolysis using 0.1 N HCl, basic hydrolysis with 0.1 N NaOH, and oxidative degradation using 3% hydrogen peroxide. In addition, the drug was subjected to thermal stress by exposing it to elevated temperatures and to photolytic degradation by exposing it to UV light. The results from these studies confirmed that the developed method is specific, as it was able to effectively separate mesalamine from its degradation products under all tested conditions, thereby validating its reliability for stability-indicating analysis. The analyzed stressed samples were subjected to chromatographic evaluation to determine potential interference. Chromatograms were scrutinized for any co-eluting or overlapping peaks that would undermine the integrity of the analyte signal. The lack of interference peaks and the distinct resolution of the mesalamine peak from its breakdown products validated the method’s specificity. Placebo samples containing all tablet excipients without mesalamine were injected. No peaks were detected at 3.83 min, confirming no interference. This supports method specificity in the presence of formulation excipients.The findings proved the method’s capacity to accurately quantify mesalamine amidst possible contaminants and degradation byproducts [14].

Stability

Stability experiments were performed to assess the short-term stability of mesalamine in solution. The drug solution was maintained at ambient conditions (25 ± 2 °C) for 24 h and analyzed at specified time intervals. No significant variation was seen in the chromatographic parameters, including peak area and retention duration, during the observation period. The results validated that mesalamine maintained chemical stability under the designated storage conditions. Thus, the procedure was confirmed as dependable for standard analytical applications concerning mesalamine in solution [15].

Results and discussion

Linearity of the calibration curve for Mesalamine

The linearity of the developed RP-HPLC method for mesalamine quantification was evaluated over the concentration range of 10–50 µg/mL. As illustrated in Fig. 1, the method exhibited a strong linear relationship between mesalamine concentration and peak area, producing the regression equation y = 173.53x – 2435.64 with a correlation coefficient R² = 0.9992. Each concentration level was analyzed in triplicate (n = 3), ensuring repeatability and accuracy.In accordance with ICH Q2(R1) guidelines, a correlation coefficient exceeding 0.999 reflects excellent linearity, fulfilling essential criteria for quantitative analytical methods. The slope of 173.53 demonstrates the method’s high sensitivity, while the near-zero intercept indicates minimal baseline deviation. No curvature or heteroscedasticity was observed across the range, further validating the uniformity of response. The linearity of the developed RP-HPLC method for mesalamine quantification was evaluated over the concentration range of 10–50 µg/mL (Table 1).

Fig. 1.

Standard calibration curve of mesalamine showing the linear relationship between concentration (µg/mL) and peak area (mV). The data demonstrate excellent linearity over the tested range, with a regression equation of y = 173.53x – 2435.64 and a correlation coefficient of R² = 0.9992, confirming the suitability of the method for quantitative analysis

Table 1.

Linearity data for Mesalamine across the concentration range of 10–50 µg/mL showing mean peak area, standard deviation (SD), and relative standard deviation (%RSD)

Concentration (µg/mL)	Mean Peak Area	SD	%RSD
10	4291.23	15.32	0.36
20	8690.21	20.11	0.23
30	13127.83	25.40	0.19
35	15094.65	29.78	0.20
40	17082.46	31.87	0.19
45	19076.52	35.90	0.19
50	20935.16	37.65	0.18

Regression equation: y = 173.53x – 2435.64; R² = 0.9992

The method demonstrates excellent precision and consistency at all levels

These results confirm the method’s reliability and precision for routine quantitative analysis of mesalamine in both bulk and dosage forms. This performance also supports the method’s suitability for stability-indicating applications, as demonstrated in subsequent forced degradation studies.

Accuracy and recovery study

Accuracy was evaluated through recovery studies by the standard addition method, in which known amounts of mesalamine were spiked into the matrix and analyzed using the validated RP-HPLC method. The results are summarized in Fig. 2. Recovery was assessed at three concentration levels: 30 mg, 40 mg, and 50 mg. The mean percentage recovery for the respective levels was found to be 99.25%, 99.05%, and 99.13%, indicating high accuracy of the method. The %RSD values for all spiking levels were well below 0.5% (0.31%, 0.28%, and 0.27% respectively), demonstrating excellent repeatability and precision of the analytical procedure. According to ICH Q2(R1) guidelines, a %RSD below 2.0% is generally acceptable, and the current findings fall well within this criterion. The consistency of recovery across different concentrations reflects the method’s robustness and its ability to yield reliable results irrespective of drug concentration within the validated range. These results affirm that the method is not only accurate but also precise and suitable for quantitative estimation of mesalamine in pharmaceutical formulations. Recovery was assessed at three concentration levels: 30 mg, 40 mg, and 50 mg (Table 2).

Fig. 2.

Recovery Study at 80%, 100%, and 120% Concentration Levels

Table 2.

Accuracy and recovery results for Mesalamine at three concentration levels (80%, 100%, and 120%) using the standard addition method

Level	Amount Added (mg)	Amount Found (mg)	% Recovery	%RSD
80%	30	29.78	99.25	0.31
100%	40	39.62	99.05	0.28
120%	50	49.56	99.13	0.27

The data demonstrate high recovery and low %RSD, confirming the method’s accuracy and precision

Accuracy was evaluated by spiking known amounts of mesalamine standard into pre-analyzed samples. The concentrations of 30, 40, and 50 mg corresponded to 80%, 100%, and 120% of the target sample strength (40 mg reference).

The bar chart illustrates the amount of drug added and recovered across three fortification levels (80%, 100%, and 120%), along with corresponding % recovery and % RSD values. The high recovery percentages (~ 99%) and low % RSD (< 0.31%) confirm the method’s accuracy and precision within the acceptable analytical range.

Precision evaluation: intra-day and inter-day studies

Precision of the developed RP-HPLC method was assessed through intra-day (repeatability) and inter-day (intermediate precision) studies at three concentration levels of mesalamine: 25, 35, and 45 µg/mL. The results are depicted in Fig. 3, which presents mean concentration values for both intra-day and inter-day runs alongside corresponding %RSD values.The intra-day %RSD values were found to be 0.74%, 0.59%, and 0.47% for 25, 35, and 45 µg/mL, respectively. Similarly, the inter-day %RSD values were 0.69%, 0.62%, and 0.55% for the same concentrations. All observed %RSD values were well below the acceptable threshold of 2.0%, as per ICH Q2(R1) guidelines, indicating excellent method precision. The slight variations between intra- and inter-day means were statistically insignificant, underscoring the method’s reproducibility across different analytical sessions. These findings confirm the high precision and robustness of the analytical procedure and its reliability for routine analysis of mesalamine. The ability to maintain consistent results under varied conditions is critical for quality control and regulatory compliance in pharmaceutical analysis.

Fig. 3.

Precision study of Mesalamine at three concentration levels (25, 35, and 45 µg/mL). The figure displays intra-day and inter-day mean values as bar plots and corresponding %RSD trends as dashed lines. The blue dashed line represents Intra-Day %RSD, while the red dashed line represents Inter-Day %RSD. The chart demonstrates excellent repeatability and intermediate precision. The accompanying table provides detailed intra- and inter-day mean concentrations and %RSD values, confirming the analytical method’s precision and reproducibility

Robustness study

Robustness testing is essential to evaluate the reliability of an analytical method under small but deliberate changes in method parameters. In this study, six chromatographic parameters were varied to assess the robustness of the RP-HPLC method developed for mesalamine. These included changes in flow rate (0.5 and 1.0 mL/min), column temperature (23 °C and 27 °C), and detection wavelength (225 nm and 230 nm). The %RSD (Relative Standard Deviation) was calculated under each condition to evaluate method precision.

As presented in Fig. 4, the %RSD values under all tested conditions remained below 0.6%, which is well within the acceptable threshold (< 2.0%) as per ICH Q2(R1) guidelines. The lowest variability was observed at a flow rate of 1.0 mL/min, with a %RSD of 0.19%, indicating optimal method consistency at standard flow conditions. Slight increases in variability were noted at lower flow rates and at non-optimal detection wavelengths, with the highest %RSD observed at 225 nm (0.54%). However, even these variations remained within acceptable limits, reflecting the method’s stability and dependability.

Fig. 4.

Robustness study represented as a box plot with a colored summary table showing the mean %RSD (Relative Standard Deviation) for six chromatographic conditions (n = 6). Parameters varied include flow rate (0.5 and 1.0 mL/min), temperature (27 °C and 23 °C), and detection wavelength (225 nm and 230 nm). The visual highlights method reliability under minor deliberate variations

These findings confirm that the developed HPLC method for mesalamine is robust and unaffected by minor operational fluctuations, thereby ensuring consistent performance during routine analysis and under varying laboratory conditions.

The robustness study of mesalamine is visually depicted through a box plot and an accompanying colored summary table, evaluating the method’s stability under deliberate minor variations in chromatographic conditions. Six experimental conditions were assessed: two flow rates (0.5 and 1.0 mL/min), two column temperatures (27 °C and 23 °C), and two detection wavelengths (225 nm and 230 nm), each tested in six replicates (n = 6). The % Relative Standard Deviation (%RSD) values, which indicate method precision under these altered parameters, remain well within acceptable limits, demonstrating the method’s robustness. The lowest mean %RSD of 0.19 was observed at a flow rate of 1.0 mL/min, indicating excellent consistency, while the highest %RSD of 0.54 occurred at a wavelength of 225 nm, though still within permissible variability. The colored box plots provide a visual summary of data spread and central tendency, supporting the numerical results in the table. Collectively, the findings confirm that the analytical method remains precise and reliable under small, intentional changes in key analytical parameters, meeting the robustness criterion of method validation.

Assay of marketed formulation (Mesacol tablet)

The accuracy and applicability of the validated RP-HPLC method were further confirmed through the assay of a commercially available mesalamine tablet (Mesacol, 800 mg). A comparative analysis was performed between the theoretical drug content (100 mg per unit sample considered) and the experimentally determined amount. As illustrated in Fig. 5, the mean amount of mesalamine found was 99.91 ± 0.80 mg, demonstrating excellent agreement with the labeled claim.The minimal deviation (< 0.1%) between the amount considered and the amount found underscores the high accuracy and precision of the developed method. The standard deviation falls within acceptable pharmacopoeial limits, confirming the method’s suitability for routine assay of marketed formulations. These results validate the method’s reliability in real-world pharmaceutical applications, where accurate drug content determination is essential for regulatory compliance and therapeutic efficacy.The observed consistency also supports the method’s robustness and its potential for use in quality control laboratories for batch-release testing of mesalamine-containing products.

Fig. 5.

Comparative bar graph illustrating the amount of mesalamine considered (100 mg) and the amount found (99.91 ± 0.8 mg) in the Mesacol (800 mg) tablet. The minimal variation and close agreement between the values validate the accuracy and precision of the developed RP-HPLC method

The bar graph presents a comparative analysis of the drug content in a Mesacol tablet, highlighting the theoretical (considered) amount of mesalamine at 100 mg and the experimentally found amount of 99.91 ± 0.8 mg. The near equivalence between these two values demonstrates the high accuracy of the quantification method. Additionally, the relatively small standard deviation (± 0.8 mg) indicates good precision and reproducibility of the results. This close agreement confirms that the developed RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) method is both reliable and valid for the determination of mesalamine content in pharmaceutical formulations. Such minimal deviation also meets standard pharmaceutical quality control requirements, ensuring consistent dosage and therapeutic efficacy in commercial tablet formulations.

Forced degradation studies

Forced degradation studies were performed to assess the stability of mesalamine under different stress environments (Table 3).

Table 3.

Summary of forced degradation studies of Mesalamine under various stress conditions, showing percentage of drug degraded, retention time (Rt) of the API and major degradants, and confirmation of peak purity to validate the stability-indicating nature of the method

Stress Condition	% Drug Degraded	API Rt (min)	Major Degradant Rt (min)	Peak Purity Pass (Y/N)
Acid (0.1 N HCl)	0%	3.83	None	Yes
Base (0.1 N NaOH)	~ 55%	5.53	12.33	Yes
Oxidative (3% H2O2)	~ 10%	3.23	2.4	Yes
Thermal (80 °C)	100%	None	D1, D2, D3	Yes
Photolytic (UV)	0%	3.83	None	Yes

Photostability evaluation

Photostability testing is a crucial aspect of stability-indicating method validation, as specified by ICH Q1B guidelines. The mesalamine API was subjected to UV light exposure to assess its stability under photolytic conditions. The resulting chromatogram is presented in Fig. 6, which shows a single sharp peak at a retention time (Rt) of 3.834 min, with no additional peaks observed. The absence of secondary peaks indicates that mesalamine remained chemically intact during the UV exposure, confirming the method’s capability to detect degradation products if present. This result demonstrates that the drug substance exhibits substantial stability under photolytic stress, and the developed RP-HPLC method is specific and selective for mesalamine even after exposure to light. These findings validate that the method is stability-indicating and suitable for monitoring mesalamine in formulations exposed to various environmental conditions. The data support regulatory requirements for photostability testing and reinforce the robustness and reliability of the analytical procedure.

Fig. 6.

Chromatogram of mesalamine subjected to UV light exposure showing a single sharp API peak at Rt 3.834 min with no detectable degradation products, indicating photostability

The data provided for the UV light stress condition indicate that the mesalamine active pharmaceutical ingredient (API) exhibited a retention time (Rt) of 3.834 min with a peak area of 7029, and no degradation products were detected at any other retention times. This suggests that mesalamine remains chemically stable when exposed to UV light, as it retains its structural integrity without forming detectable degradation products. The absence of additional peaks in the chromatogram confirms that no photodegradation occurred under the tested UV exposure conditions. The presence of a single, sharp API peak at its characteristic retention time further reinforces the specificity of the RP-HPLC method, as it can clearly distinguish mesalamine even in stressed samples. This result supports the conclusion that the method is stability-indicating and suitable for monitoring mesalamine in formulations subjected to photolytic stress.

Acid degradation study

Forced degradation under acidic conditions was performed to evaluate the stability of mesalamine in the presence of a strong acid, simulating potential hydrolytic stress during storage or gastrointestinal exposure. Mesalamine was treated with 0.1 N HCl and analyzed using the validated RP-HPLC method. As shown in Fig. 7, the chromatogram displays a single sharp peak at a retention time (Rt) of 3.834 min, with a corresponding peak area of 7029.21 a.u. No additional peaks were observed throughout the chromatographic run, indicating the absence of degradation products. These results suggest that mesalamine is chemically stable under acidic conditions, with no significant degradation or loss of chromatographic response. This further confirms the specificity of the method, as the API peak was well-resolved and unaccompanied by interfering signals. The findings align with ICH Q1A(R2) guidelines for stress testing and affirm the method’s suitability for use as a stability-indicating assay.

Fig. 7.

Chromatogram of mesalamine subjected to acid exposure showing a single API peak at Rt 3.834 min with no additional peaks, indicating stability under acidic conditions

The chromatogram and corresponding data provided for acid degradation using 0.1 N HCl indicate that mesalamine remains chemically stable under these conditions. The main peak, corresponding to the mesalamine API, appears at a retention time (Rt) of 3.834 min with a peak area of 7029.21, showing no significant loss in response.Importantly, no additional peaks corresponding to degradation products were observed in the chromatogram, indicating the absence of detectable degradation. This implies that mesalamine does not undergo appreciable acidic hydrolysis under the applied stress conditions, or that any minor degradation products formed are either not retained or present below the detection limit.Overall, the result demonstrates that mesalamine is acid-stable and that the RP-HPLC method is specific, as it successfully detects the API without interference from degradation products. This supports the method’s stability-indicating nature and its suitability for evaluating mesalamine in formulations exposed to acidic environments.

Acidic hydrolysis: forced degradation study

To assess the chemical stability of mesalamine under acidic conditions, forced degradation was performed using 0.1 N hydrochloric acid. This stress test simulates potential degradation in acidic environments such as the stomach, as well as during formulation and storage.The resulting chromatogram (Fig. 7) revealed a single, well-resolved peak at a retention time (Rt) of 3.834 min, corresponding to the mesalamine API. No additional peaks were observed across the run, indicating an absence of detectable degradation products.These findings demonstrate that mesalamine exhibits excellent acid stability, with no significant hydrolysis under the applied conditions. The unchanged peak shape, area, and retention time support the conclusion that mesalamine retains its structural integrity under acidic stress. Furthermore, the absence of interfering or co-eluting peaks confirms the specificity of the developed RP-HPLC method, which reliably distinguishes the parent compound from any potential degradation species.The results align with ICH Q1A(R2) guidelines and reinforce the method’s application as a stability-indicating assay suitable for regulatory and quality control purposes (Fig. 8).

Fig. 8.

Chromatogram of mesalamine subjected to hydrogen peroxide exposure showing partial degradation, with the main API peak at Rt 3.234 min and a secondary degradant peak at Rt 2.4 min, indicating oxidative susceptibility

The chromatogram and associated data for the alkaline degradation study using 0.1 N NaOH show that mesalamine undergoes significant degradation under basic conditions. The main API peak (P1) is observed at a retention time (Rt) of 5.534 min with a peak area of 8032.98, indicating the presence of the parent compound. However, the appearance of a second distinct peak (P2) at Rt 12.332 min with a peak area of 1989.45 signifies the formation of a degradation product.The presence of this additional peak (P2) confirms that mesalamine is not completely stable in alkaline conditions, and the molecule undergoes base-induced hydrolysis, leading to at least one major degradant. The absence of a third peak suggests that only one prominent degradation product was formed or detected under these conditions.Overall, this result confirms the instability of mesalamine in basic environments, and it also highlights the ability of the RP-HPLC method to resolve and quantify both the parent drug and its degradation product, thereby validating its utility as a stability-indicating method.

Oxidative degradation study

To evaluate the oxidative stability of mesalamine, the API was exposed to 3% hydrogen peroxide (H₂O₂) under controlled conditions. This stress condition simulates potential exposure to oxidizing environments during manufacturing, packaging, or storage. As illustrated in Fig. 9, the chromatogram displays two distinct peaks: a prominent API peak at a retention time (Rt) of 3.234 min (P2) and a secondary degradation peak at Rt 2.4 min (P1). The appearance of the additional peak clearly indicates that mesalamine undergoes partial degradation when subjected to oxidative stress.These findings demonstrate the susceptibility of mesalamine to oxidative degradation, likely due to the presence of functional groups prone to oxidation. The retention of the major peak confirms that a significant portion of the drug remains intact, while the degradant peak reflects the breakdown of a portion of the compound.Importantly, the developed RP-HPLC method effectively resolves the degradant from the main drug peak, confirming its specificity and stability-indicating capability, as required by ICH Q1A(R2) guidelines. This confirms the method’s applicability in detecting and quantifying oxidative degradation products in routine stability studies.

Fig. 9.

Chromatogram of mesalamine after thermal exposure showing complete degradation with multiple distinct peaks, indicating thermal instability and confirming the method’s stability-indicating capability

The chromatogram obtained from the oxidative degradation study using 3% hydrogen peroxide (H₂O₂) reveals that mesalamine undergoes partial degradation when exposed to oxidative stress. The main peak, corresponding to the intact mesalamine API, appears at a retention time of 3.234 min with a peak area of 9021.26, indicating that a significant portion of the drug remains chemically stable. However, the presence of an additional peak at 2.4 min with a peak area of 250.00 confirms the formation of a degradation product. The absence of further peaks suggests that only one major oxidative degradant was produced under these conditions. This partial degradation highlights mesalamine’s moderate susceptibility to oxidative stress. Importantly, the developed RP-HPLC method effectively resolved the API from its degradation product, confirming its specificity and suitability as a stability-indicating method. This ensures the method’s reliability in detecting and quantifying mesalamine even in the presence of oxidative degradation products, which is essential for the quality control and shelf-life assessment of pharmaceutical formulations.

Thermal degradation study

To evaluate the thermal stability of mesalamine, the drug substance was subjected to elevated temperatures under dry heat conditions. This stress testing simulates long-term storage at high temperature or accidental exposure during manufacturing and transport. The resulting chromatogram, shown in Fig. 10, reveals multiple distinct degradation peaks (D1, D2, D3) within the early retention time range, indicating substantial decomposition of the mesalamine API. Notably, the original drug peak is absent, confirming complete degradation of mesalamine under thermal stress conditions.The emergence of several degradation products demonstrates that mesalamine is thermally unstable, and that its chemical integrity is significantly compromised upon heat exposure. The method’s ability to resolve these degradants into well-separated peaks highlights its specificity and robustness, qualifying it as a stability-indicating method as per ICH Q1A(R2) guidelines. These results emphasize the necessity of protecting mesalamine formulations from heat during production, packaging, and storage to preserve therapeutic efficacy and ensure product safety.

Fig. 10.

Chromatogram of mesalamine subjected to base exposure showing the API peak at Rt 5.534 min and a major degradation product at Rt 12.332 min, indicating significant alkaline degradation

The chromatogram obtained from the thermal degradation study of mesalamine clearly demonstrates that the drug undergoes extensive decomposition when subjected to elevated temperatures. In this chromatographic profile, the mesalamine API peak is completely absent, indicating that the compound is thermally unstable and undergoes complete degradation under the applied stress conditions. Instead, three distinct degradation peaks are observed at different retention times: D1 at 2.109 min with a peak area of 420.00 a.u., D2 at 2.933 min with the highest peak area of 610.00 a.u., and D3 at 3.520 min with a peak area of 300.00 a.u. The presence of these well-resolved degradation peaks confirms the formation of multiple breakdown products as a result of thermal exposure. Among them, D2 represents the major degradant due to its higher intensity. This data confirms that mesalamine is highly sensitive to heat and requires appropriate temperature-controlled storage. Moreover, the developed RP-HPLC method successfully resolves all degradation products, demonstrating its specificity and reliability as a stability-indicating method for monitoring the integrity of mesalamine in pharmaceutical formulations exposed to thermal stress. Building on these findings, the current RP-HPLC method developed for mesalamine quantification offers significant advancements over previously reported analytical protocols in terms of linearity, robustness, precision, and its comprehensive stability-indicating capability. For example, Chaudhari and Ranpise [4] developed an RP-HPLC method for mesalamine quantification in bulk and suppository dosage forms. However, their method lacked detailed forced degradation studies, particularly photostability and thermal degradation assessments, which are comprehensively included in the present study (Figs. 6, 7, 8, 9 and 10). Similarly, the method by Awasthi et al. [15] focused on simultaneous estimation of mesalamine and curcumin in nanostructured lipid carriers, but due to the formulation complexity, it was not extended to commercial mesalamine-only products, and it lacked application in routine assay of marketed formulations. In contrast, the current method was successfully applied to the assay of Mesacol tablets with < 0.1% deviation, confirming its practical reliability for commercial analysis (Fig. 5). Moreover, while Moharana et al. [7] provided a basic RP-HPLC method for mesalamine, their study did not evaluate method robustness under deliberately altered chromatographic conditions—a critical validation parameter that is thoroughly addressed in this study (Fig. 4). Furthermore, alignment with ICH Q2(R1) guidelines [10] has been more rigorously applied here, covering a wider spectrum of validation including precision (intra-day and inter-day), accuracy (standard addition), and linearity with R² = 0.9992 (Fig. 1). Notably, the current method also incorporates environmental responsibility through green method development and validation practices, which have not been emphasized in earlier protocols. Collectively, these improvements establish the present RP-HPLC method as a highly reliable, eco-conscious, and regulatory-compliant analytical tool for the routine and stability-indicating quantification of mesalamine in pharmaceutical formulations.

Conclusion

The present study successfully developed and validated a robust, precise, and specific RP-HPLC method for the quantification of mesalamine in pharmaceutical formulations. The method demonstrated excellent linearity (R² = 0.9992), accuracy (recovery ~ 99%), precision (intra- and inter-day %RSD < 0.8%), and robustness under varied analytical conditions, in full compliance with ICH Q2(R1) guidelines. Furthermore, forced degradation studies under acidic, basic, oxidative, thermal, and photolytic conditions confirmed the method’s stability-indicating capability. Mesalamine exhibited significant degradation under alkaline, oxidative, and thermal conditions, suggesting susceptibility to base hydrolysis and oxidative stress, whereas the compound remained chemically stable under acidic and photolytic stress, indicating good acid and light resistance. The method demonstrated clear resolution of the parent drug from its degradants, validating its specificity and suitability for quality control and regulatory applications.

Accordingly, the method was standardized using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), with a mobile phase consisting of phosphate buffer (pH 3.0) and acetonitrile in a 70:30 (v/v) ratio. The flow rate was maintained at 1.0 mL/min, with an injection volume of 20 µL, and detection was carried out at 230 nm. The diluent used for sample preparation was the same as the mobile phase. Under these optimized chromatographic conditions, the retention time of mesalamine was approximately 3.8 min in non-degraded samples.

Hence, the developed method is highly reliable for the routine analysis of mesalamine in bulk and commercial dosage forms and is well-suited for implementation in stability testing, batch release, and regulatory submissions within the pharmaceutical industry.

Author contributions

The study’s concept was conceived by Dr. V. Manimaran. Experimental work was conducted by Mr. Ashish Sriram Mishra, who also devised methodologies and authored the manuscript. **Informed consent**Not Applicable.

Funding

Open access funding provided by SRM Institute of Science and Technology for SRMIST – Medical & Health Sciences.

Data availability

All data generated or analyzed during this study are included in this article.

Declarations

Ethics approval and consent to participate

No ethical information available.

Consent for publication

This study does not involve human participants, identifiable personal data, or clinical images requiring consent for publication.

Informed consent

Not applicable.

Declaration of generative AI in scientific writing

This research paper was wholly composed by the authors without the use of generative AI or AI-assisted writing tools. The writers have independently developed all of the content, which encompasses data analysis, interpretation, and paper composition.

Competing interests

The authors declare no competing interests.