RP-HPLC Method for Estimation of Solifenacin Succinate in API and Tablet Dosage Form

 

Bhavana V, Hemant T Kumar*, Srinivasa Y Rao, Vara Prasad K Rao

 

ABSTRACT:

A simple RP-HPLC method was developed and validated for the quantitative estimation of Solifenacin succinate in API and Tablet dosage form. In this method, mobile phase was Acetonitrile and water in the ratio of 80:20 % v/v and at a flow rate of 1.0 ml/min. The optimum wavelength for detection was 225 nm. The column used was Enable ODS reverse phase (250 mm × 4.6 mm, 5 µm particle size) C₁₈ column. The retention time of solifenacin succinate was found at 2.92 min. The linearity was observed in the range of 10–100 µg/ml with the correlation coefficient of 0.998. The proposed method was found to be simple, precise, accurate and selective and can be successfully applied for the routine analysis of Solifenacin succinate in API and tablet dosage form without any interference by the excipients.

 

 

 

INTRODUCTION:

Solifenacin succinate (Figure1) is a competitive muscarinic acetylcholine receptor antagonist. Muscarinic receptor antagonists are widely used for treatment of the syndrome of overactive bladder and urge urinary incontinence^1,2. The binding of acetylcholine to these receptors, particularly the M3 receptor subtype, plays a critical role in the contraction of smooth muscle. Solifenacin Succinate is a competitive muscarinic receptor antagonist with selectivity for the urinary bladder over salivary glands.

 

Solifenacin succinate inhibits carbachol-induced intracellular calcium mobilization more potently in bladder smooth muscle cells than in salivary gland cells. Solifenacin is approximately 98% (in vivo) bound to human plasma proteins, principally to α1-acid glycoprotein. Solifenacin is highly distributed to non-CNS tissues, having a mean steady-state volume of distribution of 600 L. Solifenacin is extensively metabolized in the liver by CYP3A4 iso‐enzyme, however, an alternative metabolic pathway is also reported.

 

IUPAC name of Solifenacin succinate is Butanedioic acid, compound with (1S)-(3R)-1-azabicyclo [2.2.2] oct-3-yl 3,4-dihydro-1-phenyl-2(1H)-iso-quinolinecarboxylate (1:1), having an empirical formula of C₂₃H₂₆N₂O₂. C₄H₆O₄ and a molecular weight of 480.55. It is freely soluble at room temperature in water, Glacial acetic acid, dimethyl sulfoxide and methanol. Various methods are available for the analysis of Solifenacin in literature like spectroscopic^3,4,5 and chromatographic methods^{6,7,8,9,10,11,12,13} for estimation of solifenacin succinate in pharmaceutical dosage form, but very few HPLC methods are reported for its estimation. So there is a need of RP-HPLC method applicable for estimation of solifenacin succinate in API and pharmaceutical dosage form. We here report a new, simple, accurate and precise RP-HPLC method for determination of assay of Solifenacin succinate in API and its tablets dosage form and validate the developed method as per ICH guidelines.

 

 

Fig. 1: Chemical Structure of Solifenacin Succinate

 

MATERIALS AND METHOD:

Instrumentation

Quantitative RP-HPLC was performed using Enable ODS reverse phase (250 mm × 4.6 mm, 5 µm particle size) C₁₈ column, a Rhenodyne injector equipped with a 20 µl sample loop, variable wavelength programmable UV detector SPD-20A VP was used. The column used was Enable ODS reverse phase (250 mm × 4.6 mm, 5 µm particle size) C₁₈ column. The HPLC system was equipped with LC solutions software.

 

Chemicals and Reagents:

Analytically pure sample of Solifenacin succinate with purities greater than 99 % was obtained as a gift sample from Rachem Pharma, Hyderabad, India and tablet formulation [Soliten] was procured from Appolo Pharmacy, Visakhapatnam, India with labelled amount 5 mg of Solifenacin Succinate. Acetonitrile (HPLC grade), water (HPLC grade), Methanol (HPLC grade) were obtained from SD Fine chemicals (Hyderabad, India), 0.45 μm Nylon membrane filters were obtained from Spincotech Private Limited, Hyderabad, India.

 

Method Development:

An RP-HPLC method was developed for the estimation of Solifenacin succinate present in tablet dosage form. The RP-HPLC method was then validated to indicate that the analytical procedure used was suitable for intended use by using various parameters like specificity, linearity, precision, accuracy, range, robustness and system suitability.

 

Selection of wavelength:

Accurately 10 mg of Solifenacin succinate was weighed and transferred into a 100 ml volumetric flask and about 25 ml of methanol (HPLC grade) was added and sonicated for 20 minutes and diluted up to the mark with methanol (HPLC grade). The resulting solution (100 µg/ml) was scanned in UV-Visible spectrophotometer from 200-400 nm to determine the λ_max against the reagent blank. The λ_max of Solifenacin succinate was found to be 225 nm.

 

Preparation of Solifenacin succinate Standard Solution:

A standard solution of Solifenacin succinate (100 µg/ml) was prepared by accurately weighing Solifenacin succinate (10 mg) and dissolved in methanol and diluted to 100 ml with methanol in the same volumetric flask.

 

Preparation of Sample Solution:

Twenty tablets were weighed and powdered. The quantity of the powder equivalent to 10 mg of Solifenacin succinate was transferred to 100 ml volumetric flask. The content was mixed with methanol (70 ml) and sonicated for 20 min to dissolve the drug as completely as possible. The solution was then filtered through a Whatman filter paper no. 41 and volume was adjusted up to the mark with methanol. An aliquot of solution (4 ml) was transferred to a 10 ml volumetric flask and the volume was adjusted up to the mark with mobile phase to obtain required concentration of Solifenacin succinate (40 µg/ml). The resulting solution was then filtered through a nylon membrane.

 

Preparation of Mobile phase:

The mobile phase consists of Acetonitrile and Water in which initially the solutions were filtered separately using 0.4 µm Nylon filter and then mixed in the ratio of 80:20% v/v. The mixture of solvents were filtered again and thoroughly sonicated.

 

Method:

The contents of the mobile phase were filtered before use through 0.45 µm membrane filter, degassed with a vacuum pump and pumped from the respective solvent reservoirs to the column at a specific flow rate. Prior to injection of the drug solutions, the column was equilibrated for atleast 30 minutes with the mobile phase flowing through the systems. Drug solutions were prepared in mobile phase. Then 20 µl of each of standard and test solutions were injected and the retention time, average peak areas were recorded. The amount of drug present in each pharmaceutical formulation was calculated by making use of the standard calibration curve.

 

Optimized Chromatographic Condition:

Column: RP C18G (250× 4.6 mm, 5 µm particle size)

Flow rate: 1 ml/min.

Detection wavelength: 225 nm

Injection volume: 20 µl.

Column temperature: Ambient.

Run Time: 6 min.

Mobile Phase: Acetonitrile: water (80: 20 v/v)

Run Mode: Isocratic.

 

In these conditions, the tailing factor was decreased, the theoretical plates and area of the peak was good where the peak was eluted around 3 min with symmetric peak shape. The optimized parameters are used for the method development and validation by HPLC.The representative chromatogram of standard and sample solution of solifenacin succinatte was shown in Fig 2 and Fig 3 respectively.

 

Fig 2 Chromatogram of standard solution of Solifenacin succinate

 

Fig 3 Chromatogram of formulation of Solifenacin succinate

 

RESULTS AND DISCUSSION:

The UV absorption maximum (λ_max) of Solifenacin succinate was fixed as 225 nm. As the final detection was made by the UV absorption spectrum, the method was validated by linear fit curve and all other parameters of this method were calculated and were discussed.

 

Method Validation¹⁴

Specificity:

The chromatogram obtained from tablet solutions were identical with that obtained from standard solution containing an equivalent concentration of Solifenacin succinate. This showed that there were no interferences from excipients. Therefore, it could be said that developed method is highly specific.

Linearity and Range:

Samples from 10-100 µg/ml were prepared from the standard stock solution. The regression line obtained was plotted. From the data obtained, correlation coefficient, slope and y-intercept were calculated and the results were shown in table 1. Ideally correlation coefficient should be not less than 0.999 and the Beer’s plot was shown in fig.4. The plot of peak area of each sample against respective concentration of Solifenacin succinate was found to be linear in the range of 10-100 µg/ml with correlation coefficient of 0.999 which meet the method validation acceptance criteria and hence the method is said to be linear.

 

Table 1. Linear regression data

Parameter	Solifenacin Succinate
Beer’s range Slope, Intercept Correlation coefficient	10 - 100 µg/mL 1753.5 4739.5 0.9986

 

Fig 4 Linearity plot of Solifenacin succinate

 

Precision:

The method precision was checked by using standard Solifenacin succinate to ensure that the analytical method was precise. Method precision is of two types:

 

Intra-day Precision:

Six injections of sample solution were taken in one day for every 3 hrs and the retention time and area of six determinations was measured and % RSD was calculated. The % RSD for six replicate injections shall be not more than 2.0 and the results were shown in table 2.

 

Inter-day Precision:

Six injections of sample solution were taken and the measure is repeated for one week in which the retention time and area of six determinations was measured and % RSD for six replicate injections shall be not more than 2.0 and the results were shown in table 2.

 

The % RSD less than 2 % in both inter-day and intra-day indicates the precision of the method.

Table 3: Precision

Precision	Intra-day*	Inter-day*
		Day 1	Day 2	Day 3
Mean Peak Area	99359	99363	99367	99872
Standard deviation	437	347.7	438.2	359.5
% RSD	0.44	0.35	0.43	0.36

*mean of six determinations

 

Accuracy:

The recovery studies carried out by adding different amounts (80 %, 100 % and 120 %) of Solifenacin succinate bulk samples to the preanalyzed sample. Percent mean recovery was calculated as shown in Table 6. The accepted limits of recovery are 98 % - 102 % and all observed data are within the required range which indicates good recovery values and hence the accuracy of the method developed. and shown in the table 4.

 

Table 4 Accuracy of method

S. No	% Level	Actual conc. (µg/ml)	Conc. Added (µg/ml)	Conc. Found (µg/ml)	Mean % Recovery* ±SD
1.	80	40	32	31.8	99.90 ± 0.081
2.	100	40	40	39.9	99.94 ± 0.047
3.	120	40	48	47.7	99.93 ± 0.035

*mean of three determinations

 

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from background levels. The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with acceptable accuracy, precision and variability. The LOD and LOQ were calculated from linear curve using formulae

 

LOD= 3.3 * σ / S

LOQ= 10 * σ / S

 

(Where σ = the standard deviation of the response and S = Slope of calibration curve).

 

LOD value for Solifenacin succinate was found to be 0.025 µg/ml and LOQ value for Solifenacin succinate was found to be 0.085 µg/ml. These data reveals that proposed method is sensitive for the determination of Solifenacin succinate.

 

System suitability:

The standard solution was prepared as per the proposed test method and injected into the HPLC system. Record the USP tailing for Solifenacin succinate peak from first injection and % RSD on replicate injections and the chromatograms were recorded. The parameters are within the acceptance limits. The results were reported in the table 5.

 

 

Table 5: System suitability parameters of Solifenacin succinate

Retention time (min)	Height	Tailing factor	HETP	Theoretical plates
2.904	50929	1.3	23.164	7369.264

 

Robustness:

The robustness of an analytical method was a 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. Robustness was done by changing the flow rate (± 0.2 ml/min), mobile phase (±5 %), changing the wavelength (± 2 nm) and the results were shown in table 6.

 

Table 6: Robustness table

Conditions	% Assay	System Suitability parameters
		Theoretical Plates	Tailing Factor
Flow Rate 0.8 mL/min	99.86	7364	1.29
Flow Rate 1.2 mL/min	99.78	7360	1.29
Mobile Phase- Acetonitrile (75): Water (25)	99.74	7366	1.30
Mobile Phase- Acetonitrile (85): Water (15)	99.68	7368	1.31
Wavelength 223 nm	99.85	7369	1.29
Wavelength 227 nm	99.79	7367	1.30

 

Estimation of Solifenacin Succinate by proposed method:

The propsed method applied to the estimation of solifenacin succinate in tablet formulation and results are shown in Table 7.

 

Table 7: Estimation of Solifenacin succinate in tablets

Sample	Labeled amount (mg)	Amount obtained by proposed method (mg)	% Mean Recovery* ± SD
SOLITEN Tablets	5	4.93	99.93 ± 0.266

*mean of three determination

 

CONCLUSION:

A reverse phase HPLC isocratic method developed has been validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity, limit of detection and limit of quantitation, for the quantitative estimation of Solifenacin succinate in tablets. The precision is exemplified by relative standard deviation of less than 2 %. A good linear relationship was observed for the drug between concentration ranges of 10 and 100 μg/ml. Accuracy studies revealed that mean recoveries were between 98 and 102%, indicative of accurate method. Accordingly, it can be concluded that the developed reverse phase isocratic HPLC method is accurate, precise and linear and therefore the method can be used for the routine analysis of Solifenacin succinate in tablets.

 

ACKNOWLEDGEMENT:

The authors are grateful to Dr.L. Rathaiah, Chairman of Lavu Educational Society for providing necessary facilities to carry out the above research work.

 

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Received on 24.05.2019                 Accepted on 21.06.2019

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Asian J. Pharm. Ana. 2019; 9(3):118-122.