Analytical Method Development and Validation of Mirabegron By Using RP-HPLC Method of Bulk and Pharmaceutical Dosage Form

The chemical name for Mirabegron is N-(4-(2-((R)-2-hydroxy-2-phenyl ethylamino) ethyl) phenyl) -2 - (2- aminothiazol-4 yl) acetamide. MIRA is used for treating overactive bladder diseases and also cardiovascular stimulation¹. Two randomized Phase I studies were conducted to evaluate the PK properties ofMirabegron².Recently, MIRA was shown to relax in-vitro human and rabbit prostate smooth muscle through activation of β3 adrenoceptor. The same group also showed that MIRA promotes smooth muscle relaxation by α1 adrenergic receptor blockade³. Excessive overconsumption of caffeine cause overactive bladder problems⁴. Peak plasma concentrations achieved at approximately 3.5 h after oral administration. Steady-state concentrations achieved within 7 days of once-daily dosing. A literature survey revealed that very few analytical methods had been reported until now for the estimation of MIRA. The majority of methods for determination of MIRA in biological fluids and pharmaceutical dosage forms include LC-MS/MS⁶, RP-HPLC^7-10, UV spectrophotometric¹¹ analytical method is available in the literature for analyzing MIRA in pharmaceutical dosage form or as bulk drug sample. So far, to our present knowledge, only one RP-HPLC method was available. So, it is necessary to develop a simple, precise, and rapid RP-HPLC method for the quantitative determination of MIRA in the tablet dosage form. This current study describes the validation parameters stated by the International Conference on Harmonization [ICH] guidelines Q2 (R1)^12-15. Fig. 1 shows the chemical structure of MIRA.

Fig. 1: Chemical Structure Of Mirabegron

MATERIALS AND METHODS

 HPLC Instrumentation and Conditions: The experiments were conducted on Agilent technologies - – Gradient system with Auto injector, Agilent C18 column (150 mm i.d.× 4.6 mm, particle size 5 μm, maintained at ambient temperature. The mobile phase consisted of methanol:0.1% OPAin the ratio 55:45 v/v. Isocratic elution at a flow rate of 0.7 ml/min at ambient temperature and using DAD detector to monitor the elute at 254nm.To determine the optimum λmax, MIRA 10 mcg /ml of working standard solution was prepared and scanned in the UV wavelength range of 200 - 400 nm utilizing MeOH as a blank. It was observed the drug showed maximum absorbance at 254 nm, which was chosen as the detection wavelength for the estimation of MIRA. The UV overlain spectraof MIRA is shown in Fig. 2. The mobile phase was filtered through a 0.45 µm nylon filter and degassed in ultra Sonicator prior to use. Preparation of Mobile Phase: Prepare mobile phase composition of methanol and acetonitrile in required ratios methanol, and 0.1% OPA were mixed in the ratio of 55:45 v/v and was filtered through 0.45 μm nylon membrane filter and degassed by sonication. Preparation of Stock and Working Standard Solutions: Accurately 5mg of pure MIRA was weighed and transferred into a 10 ml clean and dry volumetric flask, and the mobile phase was added, if necessary, sonicate to dissolve. The volume was brought up to the mark with the mobile phase. This is the primary stock solution of MIRA with a concentration of 500 μg/ml. A secondary stock solution is prepared by transfer 1ml of primary stock solution in 100 ml volumetric flask and made up the mark with a mobile phase having the concentration 10 μg/ml, again from this 5 ml is poured into 50 ml volumetric flask then the concentration would be 1 μg/ml. Further dilutions were made to obtain the concentration in the range of 5-25µg/ml of MIRA, respectively.

Fig. 2: UV Spectra of MIRA

Tablet Sample Preparation: For analysis in tablet dosage form, accurately weighed twenty tablets, and the average weight was calculated. Tablets were finely powdered, and the tablet powder equivalent to 5mg of MIRA was accurately weighed and transferred into a 10ml volumetric flask. The volume was filled up to mark with the mobile phase to the obtained concentration of 500 μg/ml and sonicate for 15 min to dissolve it. Resulting stock solution 0.1 ml was transferred to 10 ml volumetric flask and volume was make up with the mobile phase.

RESULTS AND DISCUSSION:

Optimization and Method Development: In order to acquire proper optimized HPLC conditions in the first occasion, several mobile phases, stationary phases, flow rates as well as pH of buffers were properly tested. Eventually, a mobile phase comprising of methanol and 0.1% OPA mixed in the ratio of 55:45% v/v and stationary phase made up of Agilent C18 Column with 4.6 × 150 mm, 5 µm were observed, and they are found to be utmost suitable for analyzing MIRA. The mobile flow rate and the detection wavelength were adjusted to 0.7ml/min and 254 nm, respectively at ambient column temperature. The summary of optimized chromatographic conditions for the proposed method is shown below in Table 1.

Table 1: Optimized Chromatographic Conditions for Proposed Hplc Method.

Method Validation:

Once the chromatographic and the experimental conditions were established, the method was validated by the determination of the following parameters: specificity, system suitability, linearity, precision, accuracy, robustness, the limit of detection (LOD), limit of quantitation (LOQ), solutions stability following the ICH guidelines Q2 (R1).

System Suitability (SST):

System suitability test was useful to a representative chromatogram to check the different parameters such as retention time, theoretical plates, and tailing factor. The system suitability test results for the proposed method are shown in Table 2. Thus, the system meets suitable criteria.

Table 2: System Suitability Test Result For MIRA

* = Average of 5 determinations, % RSD = percentage relative standard deviation.

Fig. 4

Fig. 4: Chromatogram of Mira Synthetic of MIRA Drug.

Specificity:

The accessibility of method specificity was determined by juxtaposing the chromatograms got from MIRA and blank solution. By mixing the most routinely utilized excipients in the mobile phase devoid of the drug. Fig. 3 and Fig. 4 shows the chromatograms of the blank as well as a synthetic drug, respectively.

Fig. 5: Calibration Graph of MIRA By

RP- HPLC

Linearity:

A series of solutions (5-25µg/ml) were prepared from the MIRA stock solution, and 20 µl of each solution was injected into the HPLC system and the peak area of the chromatogram was noted. A calibration curve was plotted by taking the concentration of the solutions on the x-axis and the corresponding peak area values on the y-axis. The calibration curves were constructed by plotting absorbance versus concentration, and the linearity was calculated by the least square regression method. A calibration curve is shown in Fig. 5. The linearity of response for the MIRA standard was estimated in the range of 5-25µg/ml. The correlation coefficient was found to be 1. Therefore, the HPLC method was found to be linear.

Precision:

The precision of the method was determined by repeatability (intra-day) and intermediate precision (interday). Repeatability was determined by performing six repeated analysis of the same working solution of MIRA on the same day, under the same experimental conditions. The intermediate precision of the method was assessed by carrying out the analysis on different days and also by another analyst performing the analysis in the same laboratory (between-analysts). It was noted that the % RSD values of precision for intra-day and inter-day precision was 0.18 and 0.14, respectively. Intra-day and inter-day % values lower than 2% lucidly, assuring that this method was found to be fairly precise and reproducible. Precision results are tabulated in Tables 3 and 4.

Table 3: Intra-Day Precision

             *Average of six determinations

Table 4: Inter-Day Precision

*Average of six determinations

Accuracy:

Accuracy of a method is defined as the closeness of a measured value to the true value. The recovery studies were carried out at 80%, 100%, and 120% levels of test concentration wereprepared and injected into the HPLC system as per methodology. Table 5 shows the accuracy results of MIRA.

Table 5: Accuracy Study

*Average of triplicate injections

Acceptance Criteria:

The % recovery values should be in the range of 98 % - 102% with % RSD NMT 2.0.

Robustness: The robustness of an analytical procedure is the measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. For the determination of a method’s robustness, parameters such as variation in detector wavelength are varied within a realistic range, and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the method’s robustness range. The absorbance was measured, and the assay was calculated for six times. The results of robustness are presented in Table 6. There were no significant changes in the chromatographic pattern when the above modifications were made in the experimental conditions, showing thus that the method is robust.

Acceptance Criteria:

 The % RSD of MIRA should be not more than 2.0 %.

Table 6: Robustness Results Of MIRA

LOD and LOQ:

Limit of Detection is the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions. The limit of quantitation is the lowest concentration of an analyte in a sample that can be determined. LOD and LOQ were obtained from the slope and the standard deviation of the intercept from three calibration curves determined by a linear regression line as defined by ICH. The limit of detection and limit of quantitation were found to be 0.0572 μg/mL and 0.1735 µg/ml respectively. Table 7 shows the results of LOD and LOQ.

Table 7: LOD And LOQ Values Of MIRA

Analysis of Miraben ER-25 Tablet Formulation:

The developed and validated method was successfully applied for the determination of MIRA in their tablet dosage form. The assay result Table 8 shows that the amount of the drug was in excellent agreement with the labeled value of the formulation. The representative sample chromatogram of MIRA is shown in Fig. 6. Table 10 represents the summary of validation parameters.

Table 8: Assay Results Of MIRA

*Average of six determinations. SD means standard deviation

Table 9: Summary of Validation Parameters