INTRODUCTION:

Solifenacin Succinate is a class of isoquinolines. The trade name of marketed solifenacin succinate is Vesicare. It is an antispasmodic drug of anticholinergic class, employed to treat the ‘overactive bladder syndrome’. It is used as a urologic agent in the treatment of involuntary urination¹.

The Solifenacin is not reported to have elevated levels of liver enzymes or liver damage. Molecular weight is 480.561g/mol. It is used in treating the overactivity of neurogenic detrusors. The cytochrome P450 system (CYP 3A4) participates in the Solifenacin Succinate metabolism. The selected drug was found to be bioavailable at about 90 percent^2-4.

Solifenacin is an antagonist of muscarinic receptors, which deals with the contractions of smooth muscles of the urinary bladder. Binding acetylcholine to muscarinic receptors (Subtype M3) enables smooth muscle contraction. Solifenacin prevents the binding of acetylcholine to these muscarinic receptors, thereby allowing the bladder to store greater amounts of urine within which the desire for urinary incontinence is decreased in turn. Solifenacin is present in human plasma, which leads to clinical activity and is metabolized extensively in the liver⁵.

Half-life removal of solifenacin is about 45 to 68 hours. Overdosing solifenacin can trigger potential outcomes. Common side effects, such as dehydration of the mouth, constipation and urinary tract infections, blurred vision, or drowsiness.

Based on the conducted literature survey, Very few methods for the quantification were developed and used for Solifenacin Succinate by RP-HPLC^6-13. Hence, we intended to develop a reliable and economical RP-HPLC method^14-19 for the determination of the selected drug Solifenacin Succinate shown in (Fig.1).

Fig. 1: Chemical Structure of Solifenacin Succinate.

MATERIAL AND METHODS:

Equipment:

The HPLC chromatographic device (WATERS) is fitted with an autosampler which is combined with a varied wavelength programmable UV detector. For the study, a reverse phase Sunfire C8 (4.6 x 150mm, 5μm) was used. LAB INDIA UV 3000 + Visible spectrophotometer with double beam UV was used to measure wavelength. Afcoset ER-200A is used to measure electronic balances.

Reagents and chemicals:

The pure pharmaceutical grade subject drug (Solifenacin Succinate) was acquired from Pharma Train Labs, Hyderabad. It has acquired the compound Potassium Dihydrogen Phosphate (KH2PO4) from FINER compound Ltd. Merck Pvt had procured the Methanol, Orthophosphoric acid, and Water. Ltd. The marketed formulation Solifenacin Succinate Dose 10 mg tablets are obtained from the local label (Mfd. by Ranbaxy Laboratories Ltd).

Optimized conditions:

Column : Sunfire C8 (4.6 x 150 mm, 5 µm)

Detection wavelength : 220 nanometers

Mobile phase Buffer : Methanol:Acetonitrile (45:45:10 v/v)

Flow rate : 1.0 ml/min

Injection volume : 30 µl

Preparation of Buffer:

1ml of trifluoroacetic acid was taken into a 1000ml flask containing HPLC water, and pH was adjusted to 3.0 with Orthophosphoric acid to get 0.1% trifluoroacetic acid.

Preparation of Mobile phase:

To prepare the mobile phase of 1000ml, 450ml (45%) of the above buffer, 450ml (45%) of Methanol, and 100ml (10%) of Acetonitrile were mixed.

Preparation of Standard solution:

25mg of the normal Solifenacin Succinate was inserted into a 25ml flask and diluted to the stage (primary stock solution). 0.6ml was also taken in a diluent-containing 10ml flask from the above stock solution (contains 60 ppm of Solifenacin Succinate).

RESULTS AND DISCUSSIONS:

Optimized method:

The optimization of the current method was carried out with an intent to develop an economical and reliable method to estimate the Solifenacin Succinate in its formulation.

Upon subsequent trials with multiple Mobile Phase compositions, the composition of Buffer: Methanol: Acetonitrile (45:45:10v/v) was selected as the appropriate mobile phase along with the Agilent column Sunfire C8 (4.6 x 150mm, 5mm) because of good resolution, plate count and symmetry of peaks noticed. Solifenacin Succinate, when determined spectrophotometrically, was found to show substantial absorbance at 220nm hence selected for the optimized method. An optimized chromatogram showing the elution of Solifenacin Succinate was shown in (Fig. 2).

Sample Preparation:

Five Solifenacin Succinate tablets are correctly measured, crushed, and transferred to 25mg Solifenacin Succinate equivalent (marketed formulation = 308.8mg tablet Powder) in a 25ml flask (Stock solution). From the above, 0.6ml was taken into a 10ml flask and diluted, which contains 60ppm of Solifenacin Succinate.

Upon the verification of the obtained chromatograms, the method was found to be specific as the elution patterns showing no interference of the excipients present.

Assay of Marketed Formulations:

The peak area responses obtained for standard and sample are evaluated and the %Assay was calculated by using the following formulae;

AT WS DT Average weight P

% Assay-----*-----*------*---------------------*-----* 100

AS DS WT Label Claim 100

A standard assay chromatogram as shown in (Fig . 3) and the values obtained were listed below.

Brand Name : Soliten

Label Claim : 10 mg

Amount Recovered : 100.55

% Amount found in Drug : 100.40%

Fig. 3: Chromatogram of Assay of Marketed Formulation.

Validation of the RP-HPLC method:

System suitability:

It is a measure to know how well the designed method was significant for determining the subject drug of interest. The details are given in (Table 1).

Table 1: System Suitability Parameters.

S. No	Parameters	Solifenacin Succinate	Specification
1	Retention Time (min)	2.9	As obtained
2	Theoretical Plates (N)	3551.15	Not less than 2000
3	Tailing Factor (T)	1.38	Not more than 2

Linearity:

To determine the linearity parameter, the series of aliquots for concentrations ranging from 20-100mg/ml are prepared. The calibration curve is plotted with ‘obtained peak area responses against the concentration’. The calibration curve for Solifenacin Succinate was shown in (Fig. 4) and the details are tabulated in (Table 2).

The method was proved to be linear for the concentrations range of 20-100μg/ml to estimate the Solifenacin Succinate with the correlation coefficient of 0.999 respectively.

Table 2: Linearity Parameters of Solifenacin Succinate.

S. No	Parameters	Solifenacin Succinate
1	Slope	2627.8
2	Y-intercept	-141.29
3	Correlation Coefficient R2	0.999
4	Regression Equation	y = 2627.8x - 141.29
5	Linearity Range	20-100 µg/mL

Fig. 4: Calibration Plot of Solifenacin Succinate.

Detection Limit (DL) and Quantitation Limit (QL):

The DL and QL are calculated from the signal to noise ratio that represents the sensitivity of the method.

DL and QL values were estimated using the formulae;

DL = 3.3 X (σ/S)

QL = 10 X (σ/S)

Where;

σ = Standard Deviation;

S = Slope.

The results of DL and QL obtained for Solifenacin are obtained as 0.93µg/mL and 3.07µg/mL.

Precision:

The parameter ‘Precision’ deals with repeatability with respect to the method and can be demonstrated by the determination of close agreement between the obtained results. Precision can be evaluated as;

1. System Precision – the six replicates are injected and analyzed on the same day using the same instrument and the same analyst.

2. Intermediate Precision - the six replicates are injected and analyzed on a different day (Ruggedness).

3. Method Precision – the six replicates of six different sample solutions prepared are analyzed to evaluate the method.

The results of System Precision, Intermediate Precision, and Method Precision were summarized in (Tables 3).

Table 3: System Precision Results

Details	System Precision Peak Areas	Intermediate Precision Peak Areas	Method Precision Peak Areas
Injection-1	152826	156293	152826
Injection-2	152093	153299	152635
Injection-3	152078	153034	152763
Injection-4	153435	154244	153722
Injection-5	152934	155261	154264
Injection-6	154964	153566	152877
Average	153055.0	154282.8	153673
Standard Deviation	1071.0	1268.1	153322.3
% RSD	0.7	0.8	656.2

The current method was proved to be precise, rugged, and reproducible for Solifenacin Succinate respectively as the % RSD obtained is meeting the acceptance criteria (Acceptance criteria: Not more than 2.0%).

Accuracy:

Accuracy is a measure of the trueness for the determined result of measurement with respect to the standard. To corroborate the reliability of the current method, the ‘% recovery’ was calculated by using the ‘standard addition method’. Accuracy was calculated from the samples to which the known concentrations of the working standard were added and % recoveries are determined & reported. Inject the standard solution in terms of known analytes such as Accuracy - 50%, 100%, and 150% standard solutions.

The percent recovery was calculated for 50%, 100%, and 150%, it was found to be 100.95%, 100.65%, and 100.05% for Solifenacin Succinate with the mean recovery of 100.55%. This indicates that the developed method was accurate.

Specificity:

It can be established by analyzing the standard against potential interferences. For Specificity Blank and Standard are injected and analyzed. From the optimized chromatogram of Solifenacin Succinate, it is evident that there is no interference and the obtained chromatogram as shown in (Fig. 2).

Robustness:

The system is said to be 'robust' as it is unaffected by changes made to chromatographic conditions such as mobile phase composition, flow rate, etc. and the percentage of RSD is measured for impact analysis. Small expected changes in the operating conditions of the optimized system are permitted and thus the robustness of the current method has been calculated to assess the effect on the method created. Reporting the percentage of RSD obtained in the (Table 4).

Table 4: Results for Robustness altering the Flow Rate & Mobile Phase Composition.

S. No	Parameters		System Suitability Results
S. No	Parameters		USP Plate Count	USP Tailing
1	Flow Rate (ml/min)	0.9	1.41	3494.29
		1	1.38	3551.15
		1.1	1.44	3466.64
2	Change in Organic Composition in the Mobile Phase	10% less	1.20	3890.88
		*Actual	1.38	3551.15
		10% more	1.49	3173.07

From the above-mentioned results, it is evident that after deliberate changes to the method with respect to the flow rate of the parameters (± 10 percent) and the composition of the mobile phase (± 10 percent), the results of the obtained system suitability meet the acceptance criteria and therefore the method was found to be robust.

Stability Studies:

‘Stability testing of new drug substances and products’, a chapter among the International Conference on Harmonization (ICH) guidelines state that the stress testing shall be conducted for an active substance to interpret its intrinsic and fundamental stability characteristics²⁰. As part of the present work, the stress degradation studies of Solifenacin Succinate shall be performed to evaluate the resistance of the drug substance towards different degradation pathways that helps in evaluating the stability conditions further.

Preparation of Stock Solution:

25mg of Solifenacin Succinate standard was weighed and dissolved (1mg/ml stock solution) into a 25ml flask containing diluent.

Acid degradation studies:

0.6ml (Stock solution) was added into a 10ml volumetric flask, 0.1N HCl (3ml) was added to this solution and the flask was kept at 60^oC for 6 hours, With 0.1 NaOH the resulting solution was neutralized. No acid degradation products were found regarding the drug in question.

Alkaline degradation studies:

From the Stock solution, 0.6ml was taken into a 10ml flask and 0.1N NaOH (3ml) was applied to this solution and maintained the volumetric flask at 60^oC for 6 hours. It neutralized the resulting solution with 0.1N HCl. There were no products of alkaline degradation noted.

Oxide degradation studies:

0.6ml of the above-prepared Stock solution was taken into a 10ml flask. Added 1ml of hydrogen peroxide (3 % w/v) and kept for 15 minutes at room temperature, and analyzed the solution. No products were found to induce oxidative degradation.

Thermal degradation studies:

The sample of Solifenacin Succinate was obtained and held for 24 hours at 110^oC. The sample solution was then prepared and analyzed. No products for Thermal Degradation have been found.

Photodegradation:

From the stock solution prepared, 0.6ml was taken into a 10ml flask and exposed under sunlight for 24 hours and analyzed. No Photodegradation products were found.

Forced Degradation Study:

'Degradation studies' suggested that the proposed approach is accurate about the results obtained for the degraded active material under the above conditions. Based on studies performed, it was found that there were no degradable compounds, so the proposed method was shown to be stable to acid, alkali, peroxide, thermal, and photolytic conditions. The results obtained for the analysis of degradation are stated in (Table 5).

Table 5: Results for Stability Studies of Solifenacin Succinate.

S. No.	Parameters	Peak Area	% of Degradation
1	Standard	152579.3	-
2	Acid	139575	8.52
3	Base	136484	10.55
4	Peroxide	141187	7.47
5	Thermal	135599	11.13
6	Photo	142982	6.29

DISCUSSION:

Based on the above study and assessment, it is evident that among all the existing methods that are employed for the estimation of Solifenacin Succinate, the current method was found reliable, effective, and minimal time-consuming in the determination of the subject drug of interest. The data retrieved for the accurate and was well-defined in the present context.

CONCLUSION:

This newly developed RP-HPLC isocratic method is found simple and reliable for the quantitative estimation of Solifenacin Succinate from bulk and its marketed formulation. The method validation was carried out according to the ICH guidelines and all the results obtained with respect to the validation parameters are found meeting the respective specifications. Hence the current method was found to be specific for the drug of interest. Also, stress-degradation testing of the drug was carried out under defined stress conditions and proved to be stable. Hence, the established method can be applied for the regular analysis in the quantification of Solifenacin Succinate.

CONFLICT OF INTEREST:

We hereby declare that there is no conflict of interest for the data imbibed in the manuscript.

ACKNOWLEDGMENT:

The authors are grateful to the Pharma Train Labs, Hyderabad for assuring the gift sample of Solifenacin Succinate and also for providing facilities and great assistance in carrying out the current research work.

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Received on 10.05.2020 Modified on 13.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3509-3514.

DOI: 10.52711/0974-360X.2021.00608