Noble Pharmacy College, "Parth-Vatika", Junagadh, Gujarat, India 362310
A stability-indicating, eco-friendly reverse-phase high-performance liquid chromatography method was developed and validated for the simultaneous estimation of Mirabegron and Solifenacin Succinate in their pharmaceutical dosage form. This fixed-dose combination is recently approved and widely prescribed for the treatment of overactive bladder. The chromatographic separation was performed using a Hypersil BDS C18 column (250 × 4.6 mm, 5 ?m). A mobile phase comprising trifluoroacetic acid buffer and methanol in an 80:20 v/v ratio was used at a flow rate of 1 mL/min, with UV detection at 258 nm. The method exhibited good sensitivity, with retention times of 1.018 minutes for Mirabegron and 1.436 minutes for Solifenacin Succinate. The method was validated across a concentration range of 5–25 ?g/mL for Mirabegron and 0.5–2.5 ?g/mL for Solifenacin Succinate. It showed excellent linearity, precision, and accuracy in accordance with ICH Q2 (R1) guidelines. Stress degradation studies demonstrated that the method is stability-indicating, as no interference from degradation products was observed at the retention times of the analytes. Degradation was within acceptable limits under acid, alkali, oxidative, photolytic, and thermal stress conditions for both Mirabegron and Solifenacin Succinate. The proposed method is simple, accurate, robust, and cost-effective, with green assessment supporting its environmental sustainability. Overall, the method is suitable for routine quality control and stability testing of Mirabegron and Solifenacin Succinate in pharmaceutical formulations.
Overactive Bladder (OAB) is a common condition characterized by an urgent and frequent need to urinate, often accompanied by difficulty controlling the urge, leading to frequent daytime and nighttime urination and sometimes involuntary urine leakage, known as urgency incontinence. Symptoms typically include sudden urges to urinate, urgency incontinence, urinating eight or more times a day, and waking multiple times at night to void (nocturia) (1). The impact of OAB extends beyond physical symptoms, often leading to emotional distress, anxiety, disrupted sleep, and a decreased quality of life that can affect social interactions and sexual activity. In women, OAB may coexist with stress incontinence, causing mixed incontinence symptoms (2,3). Management typically involves behavioral interventions such as lifestyle and dietary modifications, timed voiding schedules, and pelvic floor exercises (Kegels), alongside medical treatments when necessary. Preventative measures focus on maintaining a healthy weight, regular exercise, minimizing caffeine and alcohol intake, quitting smoking, and managing chronic illnesses (4,5).Mirabegron is a beta-3 adrenergic receptor agonist used primarily to treat overactive bladder by relaxing the detrusor muscle, increasing bladder capacity. It appears as white to off-white crystals or powder, is soluble in organic solvents like ethanol and DMSO, and has a molecular weight of 396.5 g/mol. Compared to antimuscarinics, it offers fewer side effects, making it a safer option for managing urinary urgency and frequency (6–8). Solifenacin succinate is a competitive muscarinic receptor antagonist that treats overactive bladder by blocking M3 receptors, reducing involuntary bladder contractions. It is a white to almost white powder, soluble in organic solvents such as ethanol and DMSO, with a molecular weight of 480.5 g/mol. Its selective action helps alleviate symptoms like urinary urgency and frequency, with fewer side effects like dry mouth compared to older antimuscarinics (Figure 1) (9,10).
(a)
(b)
Figure 1 Molecular structure of (a) Mirabegron and (b) Solifenacin succinate
High Performance Liquid Chromatography (HPLC) is a widely used analytical technique for separating, identifying, and quantifying components in a liquid sample. It operates by injecting the sample into a column containing a stationary phase while pumping a liquid mobile phase through at high pressure. The components of the sample separate based on their differing affinities between the stationary and mobile phases, with less affinity for the stationary phase resulting in quicker elution. HPLC is favored for its high resolution, sensitivity, repeatability, small sample requirement, and versatility compared to gas chromatography. It can be classified by scale (preparative or analytical), separation principle (such as affinity, adsorption, ion exchange, size exclusion, or chiral chromatography), and elution technique (isocratic or gradient). The two main modes of operation are normal phase chromatography, which uses a polar stationary phase and non-polar mobile phase, and reversed-phase chromatography, which employs a non-polar stationary phase with a polar mobile phase. Literature reviews indicate that no environmentally friendly design-based method exists for producing Mirabegron and Solifenacin succinate in pharmaceutical preparations. While techniques like UV, HPLC, HPTLC, and TLC are used for their estimation, they don't address sustainable production methods (11,12,21–27,13–20). Reverse phase-high performance liquid chromatography (RP-HPLC) is the most commonly used technology for pharmaceutical quality control, analyzing active pharmaceutical ingredients (APIs) and their contaminants. Traditional RP-HPLC optimization examines one component at a time, while the AQbD strategy takes a more proactive, lifecycle-wide approach to ensure product quality based on clinical performance. By replacing hazardous compounds with safe and ecologically friendly ones, green analytical chemistry (GAC) seeks to offer a multi-green analytical methodology for sustainable development (28–30). The study aimed to develop an environmentally friendly HPLC method integrating GAC principles and AQbD concepts for assessing two drugs in bulk and commercial forms. The method was verified to meet ICH Q14 requirements, ensuring reliability and eco-friendliness (31,32).
MATERIALS AND METHODS
Chemicals
Glenmark pharmaceuticals pvt.ltd gave gift samples of the usual pure medications, Mirabegron and Solifenacin succinate. A local pharmacy provided the MIRA S 50 Tablet formulation for the experiment. Water and HPLC grade methanol were bought for the investigation from Merck in Mumbai, India.
Instrumentation and chromatographic conditions
The Shimadzu HPLC LC-2010, equipped with an autosampler injector, was used for isocratic separation analyses and method development. Separation was achieved using a Hypersil BDS C18 (250 X 4.6 mm) (5μm) with a mobile phase of Trifluoroacetic acid buffer and methanol were mixed in an 80: 20 v/v % ratio, a flow rate of 1 mL/min, column temperature of 30°C, 20 µL injection volume, and detection at 258 nm.
Preparation of standard stock solution
About 50 mg of Mirabegron and 5 mg of Solifenacin succinate working standard was accurately weighed and transferred into a 100 mL volumetric flask. About 70 mL of methanol was added and sonicated for 5 minutes. Then the solution was made up with methanol. 0.1 mL of above solution was pipette out and transferred into 10 mL volumetric flask and dilute to volume with diluents. Then the solution was mixed well.
Preparation of sample solution
Twenty Mirabegron and Solifenacin succinate Tablets 50/5 mg (MIRA S 50 Tablet) were weighed and crushed. An amount of homogeneous powder with weight equivalent to 50 mg of Mirabegron and 5 mg of Solifenacin succinate was weighed accurately and mixed with 70 ml of diluent in a 100 ml of volumetric flask. The mixture was sonicated to ensure complete solubility of the drug and then made up to30 ml with diluent to get the sample stock solution containing 500 μg/ml of Mirabegron and 50 μg/ml of Solifenacin succinate and was filtered through 0.45 μ membrane filter.
Force degradation study
A method called forced degradation can be used to evaluate a substance's stability by making a pharmaceutical molecule break down under different conditions. 0.1 N HCl and 0.1 N NaOH were added to separate flasks containing the solutions of Mirabegron and Solifenacin succinate for acid and alkali degradation, respectively. After that, the volumetric flasks were kept in reflux condition at 25 °C for an hour. For the oxidation degradation study, the stock solutions of Mirabegron and Solifenacin succinate were moved into a different flask, and 30% v/v hydrogen peroxide was added. The volumetric flask was then left for three hours in a dark environment. For the purpose of thermal analysis, standard Mirabegron and Solifenacin succinate powder were baked for 24 hours at 100 °C. For 72 hours, Mirabegron powder and standard Solifenacin succinate were placed in a UV chamber and subjected to UV radiation in order to reduce the drug's photostability. To get a, all samples were diluted.
Analytical method development
For linearity, accuracy, precision, specificity, robustness, limit of detection, and limit of quantification, the developed technique was verified in accordance with ICH criteria.
Linearity
Linearity is expressed in teams of correlation co-efficient of liner regression analysis. Standard calibration curves were constructed using five standard calibration solutions in a concentration range 2.5-25 μg/ml of MIR and 0.5- 2.5 μg/ml of SOL. Each solution was chromatographed using spinchrom software and scanned between 200-400 nm and spectra were recorded. Regression statistical study on the acquired data using an MS Excel spreadsheet verified the method's linearity.
Precision
Repeatability was assessed by analyzing MIR and SOL test solutions at concentrations of 5 μg/ml and 0.5 μg/ml, respectively, with % RSD calculated. Inter-day precision was evaluated by analyzing MIR and SOL standard solutions at various concentrations (5–15 μg/ml for MIR and 0.5–1.5 μg/ml for SOL) in triplicate over different days. Intra-day precision followed the same procedure within a single day. Determine the overall data's percentage RSD.
Accuracy
The accuracy of the method was determined by calculating recovery of MIR and SOL by standard addition method. The known amount of standard solutions of MIR (5, 10, 15) and SOL (0.5, 1, 1.5) and were added to a pre-determined sample solution of MIR (10 μg/ml) and SOL (1 μg/ml). Each response was average of three determinations. The percentage recovery was calculated by measuring the absorbance and fitting these values into the regression equation of respective calibration curves.
Limit of detection (LOD) and limit of quantification (LOQ)
The LOQ and LOD were estimated using the set of six calibration curves that were used to evaluate the procedure's linearity. One might calculate the LOQ and LOD as LOQ = 10 σ/s
LOD = 3.3 σ/s
Where, σ = the SD of the response.
S = the slope of the calibration curve
Robustness
The robustness study was undertaken to examine influence of slight but deliberate variation in the chromatographic conditions. By making minor adjustments to the parameters, such as the mobile phase composition and flow rate (±0.2 mL/min), the robustness was examined.
RESULTS AND DISCUSSION
The RP-HPLC process's long-term viability is increased by combining the concepts of GAC. Consequently, we have combined all three methods to create a practical and reliable strategy. This is the concept's uniqueness, and a review of the literature demonstrated that there was no prior stability suggesting the RP-HPLC method of Mirabegron and Solifenacin succinate.
Method development studies
The analysis was conducted using a Hypersil BDS C18 column (250 × 4.6 mm, 5 μm) as the stationary phase. The mobile phase consisted of trifluoroacetic acid buffer and methanol in an 80:20 ratio, with an isocratic elution mode. The flow rate was maintained at 1 mL/min, and detection was performed at a wavelength of 258 nm using a UV detector. A 20 μL injection volume was used, with the column temperature set at 30°C. The total run time for the method was 10 minutes (Figure 2) (Table 1).
Figure 2 High performance liquid chromatography of standard telmisartan and bisoprolol
Table 1 Optimized Chromatographic condition for the estimation of telmisartan and bisoprolol
|
Parameter |
Retention time |
Tailing factor |
Theoretical Plate |
Resolution |
|
Telmisartan |
8.713 min |
1.07 |
8514.00 |
5.02 |
|
Bisoprolol |
6.496 min |
1.26 |
6750.67 |
Force degradation study
The stability of MIR and SOL was evaluated under various stress conditions. In acid degradation, drug solutions were heated with 0.1 N HCl at 60°C for 4 hours, resulting in 2.1% (standard) and 3.6% (sample) degradation in MIR, and 1.9% and 3.9% in SOL, respectively (Figure 3). Under alkaline conditions using 0.1 N NaOH at 60°C for 3 hours, MIR showed 3.9% and 3% degradation, while SOL showed 2.5% and 3.6% (Figure 4). In oxidative stress with 3% H?O? at 60°C for 2 hours, both drugs showed similar degradation as in alkali conditions (Figure 5). Photodegradation under UV exposure for 12 and 24 hours caused 3.3% and 3.9% degradation in M IR and 4.5% and 5.2% in SOL (Figure 6). Lastly, thermal degradation under the same UV exposure time showed relatively less degradation: 2.9% and 2.1% for MIR and 3.1% and 2.7% for SOL (Figure 7). These results demonstrate both drugs are susceptible to degradation under forced conditions (Table 2).
Figure 3 Chromatogram of MIR and SOL under acid degradation condition
Figure 4 Chromatogram of MIR and SOL under alkaline degradation condition
Figure 5 Chromatogram of MIR and SOL under oxidation degradation condition
Figure 6 Chromatogram of MIR and SOL under photo degradation condition
Figure 7 Chromatogram of MIR and SOL under thermal degradation condition
Table 2 Summary of force degradation of MIR and SOL
|
Stress Condition |
Content |
Area |
% Degradation |
|
0.1 N HCl |
Std MIR |
2951323 |
2.1 |
|
Std SOL |
498561 |
1.9 |
|
|
Sample MIR SOL |
2746478 503684 |
3.6 3.9 |
|
|
0.1 N NaOH |
Std MIR |
2849523 |
3.9 |
|
Std SOL |
512596 |
2.5 |
|
|
Sample MIR SOL |
2762034 505247 |
3 3.6 |
|
|
3 H2O2 |
Std MIR |
2654125 |
3.3 |
|
Std SOL |
524763 |
4.5 |
|
|
Sample MIR SOL |
2736288 502239 |
3.9 4.2 |
|
|
Photo |
Std MIR |
2869321 |
2.9 |
|
Std SOL |
498632 |
3.1 |
|
|
Sample MIR SOL |
2787642 509724 |
2.1 2.7 |
|
|
Thermal |
Std MIR |
2953962 |
3.5 |
|
Std SOL |
514852 |
3.1 |
|
|
Sample MIR SOL |
2756325 512478 |
3.2 2.2 |
Method validation studies
Linearity
The linear calibration plot for MIR in the concentration range of 5–25 μg/mL and SOL in the concentration range of 0.5 -2.5 μg/mL demonstrated good linearity, with higher correlation coefficient values of 0.999 and 0.999 for MIR and SOL, respectively (Figure 8).
Figure 8 Overlain linearity spectra of MIR (5–25 μg/mL) and SOL (0.5–2.5 μg/mL)
Precision
The peak area measurement revealed a low percentage RSD, with the range for intraday being 0.572-1.211 percent telmisartan and 0.705-1.052 percent bisoprolol, and for interday being 0.682-1.568 percent telmisartan and 0.721-1.211 percent bisoprolol.
Accuracy
Accuracy data revealed a high degree of accuracy of the devised method, with a percentage RSD value of less than 2%. Good % recovery was found in the range of 98.60-100.90 % for MIR and 98 -101.50 % for SOL at all three levels.
Limit of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ values were determined to assess the sensitivity of the analytical method. For MIR, the LOD was found to be 0.220 μg/ml and the LOQ was 0.668 μg/ml. For SOL, the LOD was 0.069 μg/ml and the LOQ was 0.209 μg/ml.
System suitability
The peak area, retention time, theoretical plate, and tailing factor of telmisartan and bisoprolol with six replicates do not significantly differ, according to system suitability data. Every system suitability parameter's percentage RSD was less than 1 %.
Robustness
By carefully altering parameters such the mobile phase composition and flow rate, the robustness was examined. All parameters' percentage RSD was found to be within the acceptable range, meaning that it was less than 2%.
Applicability of developed method
The label claim for each MIRA S 50 Tablet includes 50 mg of Mirabegron (MIR) and 5 mcg of Solifenacin succinate (SOL). The assay results showed that the mean percentage content was 99.458 ± 1.148% for MIR and 99.788 ± 1.486% for SOL, indicating that the formulation meets the required potency and content uniformity specifications.
Assessment of the environmental profile
For evaluating greenness, there are numerous techniques, including NEMI, modified NEMI, AGREE, AGREEprep, GAPI, Complex GAPI, RGBfast, BAGI, and Eco-scale. Two greenness assessment methods are applied in the presented study that can be summarized as follows:
Analytical GREEnness metric approach (AGREE)
The result of the Analytical GREEnness metric approach (AGREE) software is a circular figure that is oriented clockwise and has numbers from 1 to 12 around the outside, which represent the 12 ideologies of green analytical chemistry. The outcomes of each segment of the 12 principles were supplied together with their weighted agreement scores, which ranged from 0 to 1. The final score is represented by the core of the AGREE diagram and the net result of all 12 principles. Based on the value of the results obtained from 0 to 1, the red-yellow-green color spectrum is used. When the score values are close to 1, the color is dark green, and when they are close to 0, the color is red. The proposed method’s output score from the AGREE metrics program was 0.79. The recommended method’s score is very close to the top score of 1; hence the greenness profile was created. It is clearly mentioned in Figure 9.
Figure 9 Result of analytical GREEnness metric approach (AGREE) matrix
Green analytics procedure index (GAPI)
It is another important tool with the facility of 11 different color classifications. The particular tool is the upgraded version of “NEMI” and the term “GAPI” stands for “Green Analytics Procedure Index”. The tool can portray “hazard tolerance” and “environmental friendliness” by using and indicating different colors such as green, yellow, and red. After developing proper software “GAPI” can be used. The tool is modified in such a way; it can cover a total of 11 stages to get the proper result. It is clearly mentioned in Figure 10. The GAPI consists of 10 phases, the first of which includes solvents and reagents and the second of which includes instrument energy.
Figure 10 Result of Green analytics procedure index (GAPI) matrix
CONCLUSION
A simple, economical, selective, and robust stability-indicating RP-HPLC method was developed and validated for the simultaneous estimation of Mirabegron and Solifenacin succinate in pharmaceutical dosage forms, incorporating a green analytical approach. The method used a mobile phase of trifluoroacetic acid buffer and methanol (80:20), yielding high-resolution peaks with retention times of 21.018 min for Mirabegron and 1.436 min for Solifenacin. Validation followed ICH Q2 (R1) guidelines, confirming linearity, accuracy (98.60–100.90% for MIR and 98–101.50% for SOL), and precision (%RSD < 1.3 for both drugs across repeatability, intraday, and interday tests). LOD and LOQ values were within acceptable limits. Robustness was established with system suitability tests showing %RSD within limits. Forced degradation studies under acid, base, oxidative, photolytic, and thermal conditions confirmed the method’s stability-indicating capability, with no co-elution of degradation products. Overall, the method is suitable for routine analysis of Mirabegron and Solifenacin succinate with environmental considerations.
REFERENCES
Bansari Jagani, Dhirendra Kumar Tarai, Khyati Bhupta, Dr. Santosh Kirtane, Stability Indicating Method Development and Validation of Mirabegron and Solifenacin Succinate in Its Pharmaceutical Dosage Form by RP-HPLC with Green Assessment, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 5063-5074. https://doi.org/10.5281/zenodo.15749622
10.5281/zenodo.15749622
