New RP-HPLC Method Development & Validation for Simultaneous Estimation of Mirabegron & Silodosin as Per Ich Guidelines

Molecular Structure Of Silodosine

3. Materials And Methods

Table no 1: List of Proposed Materials:

Table no. 2: Equipments and instruments used in the study:

Optimization of Column:

SPURCIL C18 (4.6*250mm, 5µ) (DIKMA) was found to be ideal as it gave good peak shape and resolution at 1.0 ml/min flow.

Optimized Chromatographic Conditions

Equipment :          High performance liquid chromatography   equipped with Auto Sampler and PDA detector

Column                       :          SPURCIL C18 (4.6*250mm, 5µm) (DIKMA)

Buffer                          :           Ammonium acetate

PH                               :          5.0

Mobile phase              :30% buffer: 70% Methanol

Flow rate         :           1.0 ml per min

Wavelength                :           230 nm

Injection volume         :           20 l

Run time         :           10 min.

Table no 3:

3.1 Method Validation Parameters:

 3.1.1assay:

Standard Solution Preparation:

Accurately weigh and transfer 6.4mg of Silodosin and 20mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (24ppm Silodosin &75ppm Mirabegron).

Sample Solution Preparation:

Accurately weigh and transfer equivalent to 6.4mg of Silodosin and 20mg Mirabegron equivalent weight of the sample into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (24ppm Silodosin &75ppm Mirabegron).

Procedure:

Inject 10 mL of the standard, sample into the chromatographic system and measure the areas for the Silodosin and Mirabegron peaks and calculate the % Assay by using the formulae.

3.1.2. Linearity:

Preparation Of Stock Solution:

Accurately weigh and transfer 6.4mg of Silodosin and 20mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Preparation of Level – I (8ppm of Silodosin and 25ppm Mirabegron):

0.25          ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level – II (16ppm of Silodosin and  50ppm Mirabegron):

0.5            ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level – III (24ppm of Silodosin and 75ppm Mirabegron):

0.75          ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Procedure:

Inject each level into the chromatographic system and measure the peak area.

Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient.

3.1.3. Precision:

Procedure:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

3.1.4. Intermediate Precision/Ruggedness:

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day within the laboratory.

Procedure:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

3.1.5. Accuracy:

For accuracy determination, three different concentrations were prepared separately i.e. 50%, 100% and 150% for the analyte and chromatograms are recorded for the same.

Preparation Sample solutions:

For preparation of 50% solution (With respect to target Assay concentration):

Accurately weigh and transfer 3.2mg of Silodosin and 10mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents

For preparation of 100% solution (With respect to target Assay concentration):

Accurately weigh and transfer 6.4mg of Silodosin and 20mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (24ppm Silodosin &75ppm Mirabegron).

For preparation of 150% solution (With respect to target Assay concentration):

Accurately weigh and transfer 9.6mg of Silodosin and 30mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents

Procedure:

Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions.

Calculate the Amount found and Amount added for Silodosin and Mirabegron and calculate the individual recovery and mean recovery values.

3.1.6. Limit Of Detection:

Preparation of Silodosin and Mirabegron solution:

Preparation of 0.18µg/ml solution:

Accurately weigh and transfer 6.4mg of Silodosin working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent.  (Stock solution)

Further pipette 075ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Further pipette 0.15 ml of the above stock solution into a 20ml volumetric flask and dilute up to the mark with Diluent.

Preparation of 0.02µg/ml solution:

Accurately weigh and transfer 20mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Further pipette 0.3 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent

3.1.7. Limit Of Quantification:

Preparation Of Silodosin And Mirabegron Solution:

Preparation Of 0.62µg/Ml Solution:

Accurately weigh and transfer 6.4mg of Silodosin working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent.  (Stock solution) Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Further pipette 0.52ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Preparation of 0.07µg/ml solution:

Accurately weigh and transfer 20mg Mirabegron working standard into a 20ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent.  (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents.

Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Further pipette 0.9 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

3.1.8. Robustness:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

a) The flow rate was varied at 0.9 ml/min to 1.1 ml/min.

Standard solution 6.4µg/ml of Silodosin and 20µg/ml Mirabegron prepared and analysed using the varied flow rates along with method flow rate.

b) The Organic composition in the Mobile phase was varied from 63% to 77%

Standard solution 6.4µg/ml of Silodosin and 20µg/ml Mirabegron was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition in the method.

4. Results And Discussion

Trial 1

Chromatographic device        :           High performance liquid chromatography equipped with AutoSampler & PDA

Thermal profile :         Room

Packed column : spursilC8,(150×3.0mm,3m)

Mobilephase               :   60%Methanol:40%OPAPH-4

Elution rate                 :           0.9mlpermin

λmax                          :            230nm

Injection load              :           10l

Elution duration          :           10min.

Trial 2

Chromatographic device        :           High performance liquid chromatography equipped                                                                          with AutoSampler & PDA

Thermal profile          :            Room

Packed column            :PlatisilC18,(150×4.6mm,3m)

Mobile phase              :         80%Methanol:20%OPAPH-4.5

Elution rate                 :           1.0mlpermin

λmax                          :            230nm

Injection load              :           10l

Elution duration          :           10min.

Trial 3

Chromatographic device        :           High performance liquid chromatography equipped                                                                                          with AutoSampler & PDA

Thermal profile          :            Room

Packed column           :           Platisil,(250×4.6mm,5m)

Mobilephase               :         70%Methanol:30%OPAPH-4.5

Elution rate                 :           0.9mlpermin

λmax                           :           230nm

Injection  load             :           10l

Elution duration          :           14min.

System Suitability:

Equipment                  :          High performance liquid chromatography equipped with Auto Sample        and PDA detector

Column                       :          SPURCIL C18 (4.6*250mm, 5µm) (DIKMA)

Buffer                          :           Ammonium acetate

PH                               :          5.0

Mobile phase              :           30% buffer: 70% Methanol

Flow rate                     :                     1.0 ml per min

Wavelength                :           230 nm

Injection volume         :           20 l

Run time                     :                       10 min.

Figure 1: Chromatogram for system suitability

Table 4: Results of system suitability parameters

Acceptance criteria:

• Resolution between two drugs must be not less than 2.
• Theoretical plates must be not less than 2000.
• Tailing factor must be not more than 2.
• It was found from above data that all the system suitability parameters for developed method were within the limit.

4.2 Validation Parameters:

4.2.1 Assay:

Standard and sample solution injected as described under experimental work. The corresponding chromatograms and results are shown below.

Figure 2: Chromatogram for Standard

Figure 3: Chromatogram for Sample

Table 5: Results of Assay for Silodosin and Mirabegron

4.2.2 LINEARITY:

The linearity range was found to lie from 10µg/ml to 50µg/ml of Silodosin and Mirabegron and chromatograms are shown below.

Figure 4: Chromatogram for linearity-1

Figure 5: Chromatogram for linearity-2

Figure 6: Chromatogram for linearity-3

Table 6: Area of different concentration of Silodosin and Mirabegron

Figure 7: Calibration graph for Silodosin

Figure 8: Calibration graph for Mirabegron

Table 7: Analytical performance parameters of Silodosin and Mirabegron

Acceptance criteria:

Correlation coefficient (R2) should not be less than 0.999

• The correlation coefficient obtained was 0.999 which is in the acceptance limit.

4.2.3 Precision:

Precision of the method was carried out for both sample solutions as described under experimental work. The corresponding chromatograms and results are shown below.

Figure 9: Chromatogram for Precision -1

Figure 10: Chromatogram for Precision -2

Figure 11: Chromatogram for Precision -3

Table 8: Results of Precision for Silodosin and Mirabegron

Acceptance criteria:

• %RSD for sample should be NMT 2
• The %RSD for the standard solution is below 1, which is within the limits hence method is precise.

4.2.4 Intermediate Precision (ruggedness)

There was no significant change in assay content and system suitability parameters at different conditions of ruggedness like day to day and system to system variation.

Figure 12: Chromatogram for ID Precision -1

Figure 13: Chromatogram for ID Precision -2

Figure 14: Chromatogram for ID Precision

Table 9: Results of Intermediate precision for Silodosin and Mirabegron

Acceptance criteria:

• %RSD of five different sample solutions should not more than 2
• The %RSD obtained is within the limit, hence the method is rugged

4.2.5 Accuracy:

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the % recovery was calculated.

Figure 15: Chromatogram for Accuracy 50%

Figure 16: Chromatogram for Accuracy 100%

Figure 17: Chromatogram for Accuracy 150%

Table 10: Accuracy (recovery) data for Mirabegron:

Accuracy (recovery) data for  Silodosin:

4.2.6 Limit Of Detection For Silodosin And Mirabegron

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio.

Figure 18: Chromatogram of Silodosin and Mirabegron showing LOD

Table 11: Results of LOD

• Signal to noise ratio shall be 3 for LOD solution
• The result obtained is within the limit.

4.2.7 Limit Of Quantification For Silodosin And

Mirabegron

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio.

Figure 19: Chromatogram of Silodosin and Mirabegron showing LOQ

Table 12: Results of LOQ

• Signal to noise ratio shall be 10 for LOQ solution
• The result obtained is within the limit.

4.2.8 Robustness:

The standard and samples of Silodosin and Mirabegron were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

Variation in flow

Figure 20: Chromatogram showing less flow

Figure 21: Chromatogram showing more flow

Variation of mobile phase organic composition:

Figure 22: Chromatogram showing less organic composition

Figure 23: Chromatogram showing more organic composition

Table 13: Results for variation in flow for Silodosin and Mirabegron

* Results for actual flow (1.2 ml/min) have been considered from Assay standard

Table 20: Results for variation in mobile phase composition for Silodosin and

Mirabegron