Development of Chromatographic Method for The Simultaneous Estimation of Solifenacin and Mirabegron in Combination Tablet Dosage form

Overactive bladder (OAB) is a prevalent urological disorder treated with a combination of muscarinic receptor antagonists and β3-adrenoceptor agonists. Solifenacin Succinate, a selective M3 receptor antagonist, and Mirabegron, a β3-adrenergic agonist, are widely used in combination therapy for their synergistic efficacy in reducing urgency and frequency symptoms¹. Combined dosage forms of these drugs are commercially available, necessitating the development of a robust and reliable analytical method for their simultaneous estimation². High-performance liquid chromatography (HPLC) remains the preferred technique for the quantification of multiple drug components in pharmaceutical preparations due to its high accuracy, selectivity, and reproducibility³. Despite the availability of individual methods for Solifenacin and Mirabegron, limited studies have been reported for their simultaneous estimation⁴. Hence, the present study aims to develop and validate a simple RP-HPLC method for their concurrent analysis in combination tablets⁵.

MATERIALS AND METHODS

1. Procurement of Drug Samples and Chemicals:

Pure samples of Mirabegron and Solifenacin were sourced from IPCA Lab, Ratlam, M.P., India. The tablets, containing 05 mg SOL and 50 mg MIB per dose, were bought from a local market. Methanol (AR and HPLC grade) and acetonitrile (HPLC grade) were obtained from a local supplier, while HPLC-grade water was prepared at the college.

2. Identification and Characterisation of Drugs

2.1 Solubility

The solubility of all three drugs was determined by dissolving them in various solvents following IP guidelines.

2.2 Identification of drugs by their Melting Point

A digital melting point apparatus was used to determine the drug's melting point.

2.3 Identification of Drugs by IR Spectra

A drift of the drug and KBr (AR grade) was prepared and scanned between the range of 400- 4000cm-1

3.  RP-HPLC Method development for simultaneous estimation of Mirabegron (MIB) and Solifenacin (SOL).

3.1 Instrumentation

3.1.1 Shimadzu LC-IOAT HPLC Instrument

The LC system comprises a Shimadzu LC 10AT VP pump and a Rheodyne 7725 i universal loop injector with a 20 mL injection capacity. It also features a photodiode array (PDA) detector and an SPD-10 AVP UV-visible detector. The separation is performed using a Phenomenex Luna C18 column (5 mm x 50 cm x 4.6 mm i.d.). The workstation is a personal computer running CLASS-VP software from Shimadzu (Tokyo, Japan). The reservoir's volume capacity was greater than 500ml, and the mobile phase velocity was between 1 and 2 mL/min.

3.1.2 Binary pump

This is primarily beneficial on gradient runs and features with automatic changing of solvent composition- A pump supplies constant mass flow or more solvents to the arrangement (the previous one can save more components in a hypothetical arrangement)⁸. The binary pump introduces a cleaned and degassed solvent to a proportioning value. The series is built of a two-valve binary pump⁷. Solvents are measured at a percentage (chemist specified) and combined inside the pump head, where a piston of the pump meters the flow of the mixture to an outlet tube¹⁰. The outlet tubing from the pump routes the solvent flow to a sampler¹².

3.1.3 Column

The column is typically a stainless-steel tube filled with silane, octadecylsilane, or octadecylsilane-coated silica gel with a mean diameter of 3, 5, or 10 mm.

3.1.4 Photodiode array detector

The SPD-10 AVP UV-Visible detector is an advanced UV detector that monitors the entire UV spectrum simultaneously using an array of photodiodes. It detects light dispersed by a fixed monochromator across a wavelength range, providing 1nm resolution. This feature is especially useful for analyzing complex mixtures with a variety of compounds that have very different absorbance characteristics or when chromatographic peaks overlap but can be distinguished through UV absorbance¹⁴. The detector's ability to capture the full spectrum of UV absorption at a peak helps in identifying unknown substances.

3.2 Working Protocol

Product          (Label claim MIB-50 mg SOL-05 mg)

Average weight           78.5 mg.

The standard samples of Mirabegron and Solifenacin obtained for testing were evaluated for purity, with results ranging from 98% to 102%. Pure Mirabegron and Solifenacin samples were sourced from IPCA Laboratories Ltd, Ratlam, India. All chemicals and reagents used were of HPLC grade and purchased from Merck, Mumbai, India.

3.2.1 Standard stock solution Preparation

The equivalent of 10 mg each of Mirabegron and Solifenacin was accurately weighed in 100 ml volumetric flasks separately and dissolved in 25 ml of methanol to prepare standard stock solutions. After the immediate dissolution, the volume was adjusted to the mark with solvent. These standard stock solutions were observed to contain 100 mg/ml of Mirabegron and Solifenacin

3.2.2 Selection of sampling wavelengths

10 mg of Mirabegron and Solifenacin were carefully weighed separately in 100 ml volumetric flasks. After they dissolved completely, the volume was adjusted to the mark with solvent. The resulting standard stock solutions contained 100 mg/ml of Mirabegron and Solifenacin. Working standard solutions with a 5 mg/ml concentration were prepared by diluting the stock solutions appropriately (Donald). mg//ml of each drug was scanned in the range 200- 400 nm in the spectrum mode at the low scan speed to obtain the overlain spectra of these drugs.

3.2.3 Selection of mobile phase and optimization of method -

Different column chemistries, solvent types, solvent strengths (vol. fraction of organic solvents in the mobile phase, pH of the buffer), detection wavelengths, and flow rates were tested to identify the chromatographic conditions that offer the best separation. The mobile phase conditions were fine-tuned to ensure that the components do not interfere with the solvent and excipients. Additional criteria considered include the analysis time. The k values of eluted compounds were also evaluated to ensure optimal separation. Peaks, assay sensitivity, solvent noise, and the use of the same solvent system for extracting drugs from formulation matrices during drug analysis were also considered¹⁷. The same aqueous mobile phases containing methanol, acetonitrile and water were also evaluated¹⁴. The three aforementioned solvents were found to give the best results. The technique was further optimized by varying the mobile phase concentration, and the results are then presented¹⁵. The study found that the highest resolution regarding peak symmetry, resolution run time and other parameters was separated by a 55:30:15 (v/v) ratio mixture of methanol: acetonitrile: water as mobile phase. It determines flow rate by evaluating the impact of various pressures on peak area and resolution, which was found to be a 0.6 ml/min optimum¹⁸.

3.3 Optimized chromatographic conditions

The optimized chromatographic conditions are reported in Table 1.

Table 1: Optimised chromatographic conditions

4.1 Assay of SOL and MIB individually and in combination

4..1.1 Standard stock solution Preparation

Methanol was used as a common solvent for these drugs. 05 mg each of SOL and MIB were accurately weighted and then dissolved in 50 ml of solvent to achieve a solution of 1000mg/ml

4.1.2 Standard solution Preparation for the linearity study

From the respective standard stock solution of 1000 mg/ml, different dilutions were made for each individual drug, with concentrations given in Tables 5.6 and 5.7 along with the respective solvent. Afterward, 20 ml of these solutions were injected into the LC system with a Hamilton syringe. The chromatograms were then recorded at 239 nm.

4..1.3 Analysis of mixed standard

20mL solutions of these different mixed standard solutions of known concentration from the standard stock solutions of 1000 mg/ml of the drugs were injected by Hamilton syringe into the LC system, respectively, and then their chromatograms were recorded. After that, we calculated the concentration of individual drugs by extrapolating the value of the area from their calibration curves, respectively.

5.1.2 Analysis of the tablet

Moreover, the individual standard analysis's satisfactory result was applied to the quantitative study of all the commercially available tablet drugs. For the stock solution of tablet dosage form, 20 tablets were weighed, and the average weight was obtained. And then they were ground to get fine powder. Then, powder equivalents to 10 mg of SOL (from the respective quantity of MIB) were taken into the standard 50ml volumetric flask and dissolved in 30ml of methanol with vigorous shaking for 5- 10 minutes. The supernatant liquid was transferred to a 100 mL volumetric flask through a Whatman #41 filter paper. The obtained residue was washed twice with solvent, and the combined filtrate was made up to the 100ml mark. 10 ml of the above solution was then diluted to 100 ml with solvent. The sample solutions of the required concentrations of all three drugs were prepared in six replicates. An aliquot of 20uL of each replicate was injected into the system, and their chromatograms were recorded.

5.2 Validation of Developed Method

5.2.1 Linearity

The linearity of analytical procedures is the ability (within the limits) to obtain test results directly proportional to the amount of analyte in the sample. Five different concentrations were analysed, and for each concentration, the area was measured three times. The mean area was taken, which was incorporated into the calibration plot. We determined the regression equation, correlation coefficient, and standard calibration curve of the drug.

5.2.2 Accuracy (Recovery study)

Per ICH guidelines, a recovery study was performed to check the accuracy of the developed method. To analyse a purified chromatographed sample, standard solutions of all the drugs corresponding to 80, 100, and 120% of their drug content were added. The recovery study was then repeated.

5.2.3 Repeatability Precision

The repeatability was done for five replicates at five concentrations in the linearity range for SOL and for MIB, representing the precision under the same functioning condition over short intervals of time.

5.2.4 Intermediate precision

Precision was studied for intra-day and inter-day precision. Inter-day precision is the precision study carried out on different days, and intraday precision is the precision study carried out on the same day at various time intervals by the same solution. Here, six replicate samples of solutions were prepared from the stock solution. The drugs were determined for an intra-day precision study at one hour, three times on the same day. Method of Determining the Amount of Drug Contents In the first, second, and third inter-day studies, the amount of drug content was calculated on three different days.

 RESULTS

Solubility of Selected Drugs

Table 2: Solubility of drug in different solvents

Melting points of Selected Drugs

Table 3: Melting Point of Drugs

6.1 IR Spectra of the Selected Drugs

6.1.1 Solifenacin

The IR spectra of pure API Solifenacin are given as under in Figure, with wavenumber interpretations enlisted in Table

Fig. 4: FT-IR Spectra of Solifenacin

IR interpretation of Solifenacin

6.1.1 Mirabegron

The IR spectra of pure API Solifenacin are given as under in Figure 6.2, with wavenumber interpretations enlisted in Table 6.4

Fig. 5: FT-IR Spectra of Mirabegron

: IR interpretation of Mirabegron

Overlain spectra of Selected Drugs

The UV overlaying spectra of both the selected drugs was measured. It was obtained as shown in figure 6.3

Fig 6.3: Overlain spectra of Mirabegron and Solifenacin

2.1 Calibration plots of both drugs - individually and in combination

The chromatogram clearly showed that SOL relented at time 4.918 min (fig. 6 .4) and MIB at 3.256 min (fig. 6.6). The area was measured, and the calibration curve was drawn between the peak area and their concentrations. The calibration curve clearly showed that SOL and MIB have linearity ranges of 5-70 mg/ml, respectively (fig. 6.5 & 6.7).

Table 6.5: Linearity of SOLIFENACIN

Fig 6.4: Chromatogram of SOL

Fig 6.5: SOL Calibration curve

Table 6 : Linearity of MIB

Fig 6.6: Chromatogram of MIB

Fig 6.7: MIB Calibration curve

Table 7: Analysis of mixed standard

7.1 Evaluation of Tablet formulation

The chromatograms showed that SOL and MIB were eluted at 4.918 and 3.256, respectively. Using the area, the concentrations of these drugs were referred to their calibration curves. The results and their statistical results are shown in Table 6.8

Chromatogram of MIB and SOL in tablet sample

Table 8: Result analysis and statistical validation of Tablet

Validation of Developed Method

Linearity

Figures 6.10 and 6.12 show the drug's standard calibration curve. Using the mean of the The response ratio (response factor) is calculated based on the observed AUC and the corresponding concentration value. calculated by dividing the AUC by the respective concentration.

Table 9: Linearity of SOL

Table 10: Response Ratio Data for Linearity of SOL

Fig 6.9: Response Ratio Curve of SOL

Fig 11: Solifenacin Linearity test curve

Table 12: Linearity of MIB

Table 13: Response Ratio study for Linearity of MIB

Fig. 6.11: Response Ratio Curve of MIB

Fig 14: Linearity test curve of MIB

Accuracy Study

Results and statistic outcomes of accuracy study are shown at Table 6.13 and 6.14.

Table 15: Result of recovery study

Table 16: Result of statistical validation of recovery study

Repeatability Precision

Table 14 and 15, presents respectively results of repeatability.

Table 14: Repeatability of SOL

Table 15: Repeatability of MIB

Intermediate Precision - The results of inter- and intra-day precision study are shown under. Table 6.17 shows the intra- day precision results along with their statistical validation. Table 6.18 shows the inter-day precision results along with their statistical validation.

Table 16: Result and statistical validation of intra-day precision study

Table 17: Result and statistical validation of inter-day precision study