A Robust RP-HPLC Method for Silodosin in Bulk and Capsule Dosage Form: An Innovative Perspective on Stability Determination

Sandip Sen¹, Mitta Chaitanya², Bairam Ravindar³, Kalepu Swathi², Rajasree Suram⁴, Ameeduzzafar Zafar⁵, Sowmya Perudi³

¹Department of Pharmaceutical Chemistry, School of Pharmacy,

Guru Nanak Institutions Technical Campus, Ibrahimpattnam, Hyderabad - 501506, Telangana, India.

²Department of Pharmaceutical Chemistry,

Bojjam Narasimhulu Pharmacy College for Women, Hyderabad - 500059, Telangana, India.

³Department of Pharmaceutical Analysis, Srikrupa Institute of Pharmaceutical Sciences,

Velikatta, Kondapakka, Siddipet - 502277, India.

⁴Department of Pharmaceutical Analysis,

Gyana Jyothi College of Pharmacy, Hyderabad - 500098, Telangana, India.

⁵Department of Pharmaceutics, College of Pharmacy, Jouf University, Sakaka 72341, Al-Jouf, Saudi Arabia.

*Corresponding Author E-mail: sandipsen2010@gmail.com

ABSTRACT:

Silodosin, an α-adrenoreceptor antagonist primarily prescribed for benign prostatic hyperplasia. The present investigation was aimed to establish and validate an RP-HPLC method to ensure stability assessment, applicable for quantifying the substance in bulk as well as capsule formulations. The USFDA approved this medication in August 2008 due to its high uroselectivity, specifically targeting the prostate. The RP-HPLC method was developed using a mobile phase Acetonitrile: Triethylamine buffer pH 4.6: Methanol (70:20:10) at a flow rate of 1ml/min. The retention time was 2.44 min. The validation procedures adhered to ICH guidelines. The method exhibited a linear response within the 10-50µg/ml range, demonstrating a regression coefficient of 0.999. The average percentage assay was determined to be within the range of 99.00-99.9%, validating the method. The percentage relative standard deviation (%RSD) remained below 2%. Stress studies revealed no noteworthy alterations in drug retention times, and additional peaks were observed during degradation conditions. This validated RP-HPLC method is deemed suitable for routine analysis and quantification of Silodosin in both its bulk and capsule dosage, providing a reliable method to determine drug content and degraded products.

KEYWORDS: Silodosin, RP-HPLC, ICH-Guidelines, Method Validation.

INTRODUCTION:

Silodosin, depicted in Figure-1, chemically known as 1-(3-hydroxypropyl)-5-[(2R)-({2-[2-[2-(2, 2, 2-trifluoroethoxy) phenoxy] ethyl} amino) propyl] indoline-7-carboxamide. A selective third-generation α1A adrenergic receptor antagonist approved by the FDA in 2008¹.

Its primary therapeutic emphasis is centered on alleviating the manifestations and indications of benign prostatic hyperplasia through targeting prostate and urinary bladder². It acts as muscle relaxant for lower urinary tract by counteracting the α1A adrenergic receptor³.

Several analytical methods have been developed and validated to quantify Sildosin in pharmaceutical or clinical samples, as found in the literature review. In 2014, Jahan and Malipatil et al. presented a UV spectrophotometry method⁴. Similarly, spectrofluorimetry method introduced the in 2013⁵. More sensitive approaches, such as high-performance liquid chromatography (HPLC)^6-8 and high-performance thin-layer chromatography methods also reported⁹. Additionally, ultra-high-performance liquid chromatography (UHPLC) methods^10-11 and liquid chromatography-tandem mass spectrometry¹²alongside electrochemical sensing methods introduced were developed and validated¹³.

The focus of this study was to develop and validate a cost-effective stability-indicating RP-HPLC method for determining silodosin in both bulk and capsule dosage forms under various stressed conditions.

Fig1. Chemical structure of the Silodosin

MATERIALS AND METHODS:

Material:

Silodosin was sourced from Aurobindo Pharma Pvt. Ltd, based in Hyderabad, India. HPLC-grade methanol, acetonitrile, and triethylamine were procured from S.D. Fine Chemicals, located in Mumbai, India.

Chromatographic Conditions:

Choosing the right method relies on various factors such as the sample's characteristics, polarity, molecular weight, pKa value, and solubility. The Silodosin used in this study exhibits a polar nature, hence a reverse-phase high-performance chromatographic method (RP-HPLC) was employed. The experiment utilized a cyber lab LC-100 HPLC system equipped with semi-preparative, and quaternary low-pressure gradient capabilities, configured in both isocratic and gradient modes. Detection was facilitated using a UV detector. Elution was carried out through a C18-3V column (250mm×4.6 mm, CYBER Sil) at 30°C, with data interpretation performed using DS-100 chromatographic workstation software. The mobile phase composition comprised Acetonitrile: Triethylamine buffer at pH 4.6: Methanol in a ratio of 70:20:10% v/v, flowing at 1ml/min. To achieve a stable baseline, the mobile phase ran for an initial duration of 60min. Subsequently, 10µl of Silodosin working standards were injected, and the respective chromatograms were recorded. The elution process lasted for 5 minutes.

Preparation of mobile phase:

The mobile phase was prepared by combining acetonitrile, triethylamine buffer adjusted to pH 4.6, and methanol (70:20:10% v/v). Sonicate for 15 minutes to remove dissolved gases.

Preparation of standard stock solution:

Exactly 10mg of Silodosin was placed into a clean, dry 10ml volumetric flask, dissolved in 10ml of the mobile phase (1000µg/ml). The sample was sonicated for removal of any trapped gases.

Preparation of working standards:

Using the previously prepared stock solution, volumes of 0.1, 0.2, 0.3, 0.4, and 0.5ml were withdrawn and diluted with a suitable diluent upto 10ml to get concentration range from 10 to 50µg/ml.

Validation Parameters:

The method proposed underwent thorough validation concerning specificity, linearity, accuracy, precision, robustness, and ruggedness following the guidelines outlined by ICH¹⁴. The %RSD was calculated for all these parameters.

Preparation of test solutions:

100mg of the Silodosin working standard was dissolved in 100ml mobile phase and sonicated for 30 mins. From this stock, solution 0.1, 0.2, 0.3, 0.4, and 0.5ml were withdrawn and transferred into 10ml volumetric flasks, followed by volume adjustment to 10ml using the same solvent. This process generated concentrations ranging from 10 to 50µg/ml¹⁵.

Linearity:

A linearity assessment was conducted using 10-50 µg/ml standard solutions. Injection of 20µl from each standard solution occurred into the HPLC column system following specified conditions and calibration curve was plotted¹⁶.

Precision:

To assess method precision, the assay of capsule dosage was conducted at intra-day and inter-day at three different concentrations 10, 30, and 50µl/ml respectively. The %RSD (relative standard deviation) of the obtained results was calculated¹⁷.

Accuracy (Percentage Recovery Studies):

The recovery study was carried out by standard addition method. For the recovery studies, three dilutions were prepared utilizing both standard drug and commercially available formulations at 50%, 100%, and 150% levels¹⁸.

Robustness:

Robustness analysis involved variations in different conditions, such as alterations in column temperature (i.e., room temperature and 40°C) and changes in flow rate. The 20µg/ml solution was chosen for the robustness study¹⁹.

Limit of Detection and Limit of Quantification:

The determination of LOD and LOQ relied on visual evaluation, considering the Signal-to-Noise (S/N) ratio applicable to the procedure, indicating baseline noise. A comparison between a low concentration of the analyte and a blank was made. Using the Standard Deviation of the response and slope, LOD and LOQ were calculated using the provided equation²⁰.

LOD= 3.3 σ/S

LOQ=10σ/S.

The slope of the calibration curve

σ = Standard deviation of peak and

S= Slop of calibration curve

Forced degradation and stability-indicating tests^21,22:

A stock solution was created by dissolving 10mg of standard silodosin in a 25mL mixture of acetonitrile, triethylamine buffer at pH 4.6, and methanol (in a ratio of 70:20:10% v/v) via sonication for 10minutes. The degraded solution was filtered using a 0.20μm PTFE syringe filter prior to analysis.

For Acidic Degradation:

In the presence of IS, 2.5ml of 1.0M HCl was added to 7.5ml of the stock standard solution. The mixture underwent reflux at 55°C for 1hour, followed by cooling and neutralization to pH 7.0 using 1.0M NaOH. Subsequently, 6.25mL of the solution was diluted to 25 mL with ultrapure water. The resultant solution was filtered through a 0.20μm PTFE syringe filter and then analyzed using the RP-HPLC method.

For Alkaline Degradation:

In the presence of IS, 2.5ml of 1.0M NaOH was added to 7.5ml of the stock standard solution. This mixture underwent reflux at 55°C for 1hour, followed by cooling and neutralization to pH 7.0 using 1.0M HCl. Afterwards, 6.25mL of the resulting solution was diluted to 25mL with ultrapure water. Subsequent to filtration through a 0.20μm PTFE syringe filter, the solution was analyzed using the RP-HPLC method.

For Oxidative Degradation:

In the presence of,, 2.5ml of 5% H2O2 was added to 7.5 ml of the stock standard solution. This mixture underwent reflux at 55°C for 1hour, followed by cooling and adjustment to a final volume of 25mL using ultrapure water. Finally, the solution was filtered through a 0.20μm PTFE syringe filter and subjected to RP-HPLC analysis.

For Thermal Degradation:

10mg of silodosin powder underwent exposure to 80°C for 24hours. Subsequently, each powdered sample was dissolved in a previously mentioned identical mixture, aiming to reach a SIL strength of 50ppm. The resulting solution was then filtered through a 0.20μm PTFE syringe filter.

Estimation from Capsule Dosage Form:

The assay was carried out using 20 Silodal-8 capsules by Sun Pharma, each containing 8 mg of silodosin. 100mg of silodosin powder was placed into a 100mL volumetric flask, and 70mL of diluent was added.. Sonication for 30minutes was conducted, and then filtration through a membrane filter took place. 10mL of the stock solution was diluted to 100mL with diluent, resulting in a concentration of 100µg/ml. 3ml of this solution was diluted to 10ml to get concentration of 30 µg/ml and used for the assay. Concentrations were determined using the direct comparison method formula, and the retention time was noted.

C1 A1

---------- = ---------

C2 A2

C1: Concentration of standard solution of silodosin

C2: Concentration of test sample solution of silodosin capsule

A1: Peak area of a standard solution of sildosine

A2: Peak area of test sample solution of silodosin capsule

Statistical Analysis:

The data were analyzed using Graph Pad Prism 5 (GraphPad Prism 5.00.288). The data were presented as mean and Standard deviation (SD).

RESULT:

HPLC method development and optimization:

RP-HPLC emerges as a rapid, precise, and sensitive technique for sample identification and quantification. Employing a mobile phase composition of Acetonitrile: Triethylamine buffer pH 4.6: Methanol (70:20:10 v/v) with a flow rate of 1ml/min and a column temperature of 30°C facilitated successful drug separation. Detection at 267nm using a UV detector (Fig-2) highlighted a retention time for sildosine at 2.443min within a total 5 minutes runtime, as depicted in Figure 3.

Method Validation:

Linearity:

Within the 10-50µg/ml concentration range, a well-correlated calibration curve was observed using the least-squares linear regression method. The regression equation (y=39488x+21287) displayed an r²= 0.9991 (Figure-4). The % RSD values below 2% in Table 1 indicated robust correlation and method adequacy.

Figure-4: Calibration graph of Silodosin by RP-HPLC method

Table 1: Showing the result of the linearity plot

Parameter	Result
Linearity range	10-50µl/ml
Slop	39488
intercept	21287
Regression coefficient	0.9991

Precision:

Precision assessments for both interday and intraday variations at concentrations of 10, 30, and 50 µg/ml were detailed in Table 2. The %RSD observed ranged from 0.75-1.78 % for interday and 0.77-1.62 % for intraday precision.

Table 2: Inter-day and intra-day precision data of the optimized method

Concentration (µg/ml)	Interday		Intraday
Concentration (µg/ml)	Concentration found (µg/ml)	RSD (%)	Concentration found (µg/ml)	RSD (%)
10	9.83±0.12	1.22	9.87±0.16	1.62
30	29.76±0.53	1.78	29.83±0.23	0.77
50	49.27±0.37	0.75	49.54±0.52	1.05

Recovery Study (%):

The recovery study involved augmenting 25%, 50%, and 125% additional concentration to the 20µl/ml of sildosine, as illustrated in Table 3. Recovery values ranged between 99.87±0.51% and 100.8±0.47%, all within acceptable limits.

Robustness:

Evaluation of robustness under varied conditions (flow rate, temperature) in Table 4 revealed % RSD values below 2% for the peak area and retention time across different conditions, confirming a robust method.

Table 3: Accuracy as the recovery of Silodosin after the addition of extra drug

Level	The initial amount presents in the sample (µg)	Amount added (µg)	Total Amount (µg)	Amount Found (µg)	% Recovery	Mean± SD (%)	%RSD
25	20	5	25	25.19	100.8	100.8±0.40	0.11
				25.95	101.2
				25.46	100.4
50	20	10	30	31.46	100.0	99.87±0.51	0.51
				31.58	100.3
				31.57	99.3
125	20	25	45	45.83	101.8	101.4±0.58	0.57
				45.33	100.7
				45.73	101.6

Table 4. Robustness data of Silodosin at a different conditions

Robustness at different flow rates
Serial No	0.8ml /min at 30°C		1ml/min at 30°C		1.2 ml/min at 30°C
Serial No	Area	Retention time	Area	Retention time	Area	Retention time
1	842926	2.865	842946	2.443	842901	2.065
2	842929	2.841	842966	2.412	842911	2.063
3	842920	2.852	842976	2.451	842901	2.062
Mean	842925	2.853	842963	2.435	842904	2.063
SD	4.583	0.012	15.28	0.0206	5.774	0.0015
% RSD	0.0005	0.421	0.0081	0.84	0.0006	0.074
Robustness at different flow rates
S.N	1ml /min at 25°C		1ml/min at 30°C		1ml/min at 35°C
1	842011	2.921	842986	2.453	842070	2.156
2	843016	2.91	842946	2.459	842060	2.141
3	843123	2.92	842916	2.432	842026	2.123
Mean	842717	2.917	842949	2.448	842052	2.140
SD	613.5	0.0060	35.12	0.01418	23.07	0.01652
% RSD	0.073	0.21	0.0042	0.57	0.0027	0.77

Limit of Detection and Limit of Quantification:

Using the signal-to-noise method, the Limit of Detection was determined at 1.7µg/ml, and the Limit of Quantification at 5.3µg/ml.

Stress Degradation Study:

Post RP-HPLC method development, stress degradation studies under diverse conditions (acidic, alkaline, oxidative) detailed degradation products at 2.10-3.80 min across all stress conditions (Table 5, Fig. 5). The reported method was able to identify the impurity peaks arised due to applied stress conditions.

Figure 3: RP-HPLC chromatograms depicting silodosin under varied applied stress conditions including acid degradation (A), alkali degradation (B), oxidative degradation (C), and heat degradation (D).

Estimation of Silodosin from Capsule Dosage Form:

The assessment of the capsule dosage form was performed through a standard comparison method. This method, established using the RP-HPLC technique on a purchased capsule formulation, yielded an average percentage assay of 99.86% (Table 6). The assay outcome confirmed the drug's presence as per the labeled claim, suggesting the potential applicability of this method for estimating other dosage forms.

Table 5. Results of the Forced Degradation studies

Stressed Conditions	Heat temp (°C)	Time (h)	Percentage recovery study by RP-HPLC method at 267nm
Stressed Conditions	Heat temp (°C)	Time (h)	Drug recovered (%)	Drug decomposed (%)
Standard Drug	-	-	100	-
Acid hydrolysis 0.1N HCl	55	1h	91.11	9.89
Alkaline hydrolysis 0.1N NaOH	55	1h	99.09	0.91
Oxidative degradation 10% H2O2	55	1h	94.17	6.83
Heat Degradation	55	1 h	98.99	1.01

DISCUSSION:

Several single analytical stability-indicating RP-HPLC methods were reported for silodosin and its impurities. The previous studies have developed RP-HPLC methods with different compositions of organic solvents, and buffers like methanol, acetonitrile, and water, and in most of the cases they used ortho-phosphoric acid or phosphate buffer. The study utilized a mobile phase consisting of acetonitrile and 5mM ammonium acetate in a ratio of 90:10% v/v. This mobile phase was maintained at a constant flow rate of approximately 1.2 mL/min²³. The development and validation procedures were performed using a detection wavelength of 229 nm. The method exhibited outstanding linearity, with a correlation coefficient (r2) close to 0.998, within a linearity range of 8-18μg/mL for silodosin. While the method is sensitive, it presents a narrow linearity range with a slightly lower correlation coefficient. The percentage recovery achieved was 99.97%. For the validation parameters % RSD was found to be less than 2. However, the reported method did not approach the stability-indicating assay method.

This study aimed to develop a method for accurately determining related substances and degradants that is both cost-effective and rapid. Multiple experiments were conducted using a UV-visible detector, varying pH levels, flow rates, and column temperatures. Different combinations of buffers and organic solvents such as acetonitrile and methanol were explored. However, the use of buffer with a small amount of organic phase resulted in peak asymmetry, with tailing exceeding the ICH recommendation of less than 1.5. To address this issue, trials were conducted using triethylamine as a modifier. The best results were obtained with a mobile phase composition of Acetonitrile: Triethylamine buffer pH 4.6: Methanol (70:20:10v/v) with a flow rate of 1ml/min and a column temperature of 30°C facilitated successful drug separation. Within the 10-50µg/ml concentration range, a well-correlated calibration curve was observed using the least-squares linear regression method. The %RSD values below 2% indicated a robust correlation. Both interday and intraday variations at concentrations of 10, 30, and 50µl/ml validate the method's accuracy and precision with %RSD values below 2%. The recovery study values ranged between 99.87±0.51% and 100.8±0.47%, all within acceptable limits. %RSD values below 2% at each level testified to the method's precision and optimization. Similarly, the robustness under varied conditions (flow rate, temperature) revealed %RSD values below 2%. Stress degradation studies under diverse conditions (acidic, alkaline, oxidative) were carried out. Notable changes under acidic and oxidative conditions suggested superior stability in alkaline conditions. The assay on marketed capsule formulation yielded an average percentage assay of 99.86%.

The stability-indicating assay method was developed under acidic, alkaline conditions at 55°C. The thermal degradation was carried out at 55°C. The reported method was able to detect notable changes under different stressed conditions suggesting superior stability in alkaline conditions. The study indicated degradation beyond 55°C and highlighted stability within the pH range of 7.1-8.2 for oral liquid and parenteral formulations, advising storage under standard cool and airtight conditions to prevent thermal degradation.

CONCLUSION:

An economical, fast, precise, and validated method was established for analyzing silodosin and its degradants. Utilizing a mobile phase consisting of Acetonitrile, Triethylamine buffer (pH 4.6), and Methanol in a ratio of 70:20:10 v/v, with a flow rate of 1mL/min and a column temperature of 30°C, effectively separated the drug within a concentration range of 10-50µg/mL. The validation parameters exhibited % RSD values below 2% as per ICH guidelines. The reported method was able to detect degradants under different stressed conditions. The reported method can be used for routine analysis of both bulk drugs and degraded products within capsules and other dosage forms.

CONFLICT OF INTEREST:

There is no conflict of interest.

ACKNOWLEDGMENT:

The authors are also thankful to the School of Pharmacy, Guru Nanak Institutions Technical Campus, and Srikrupa Institute of Pharmaceutical Sciences (Telangana, India) for providing the necessary facilities for research work.

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Received on 13.04.2024 Revised on 15.11.2024

Accepted on 02.04.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5170-5176.

DOI: 10.52711/0974-360X.2025.00746

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No	Concentration of Standard	Peak Area of Standard	Peak Area of Sample	Concentration Found	% Purity	Average
1	30	1267247	1278537	30.2	100	99.86%
2	30	1271653	1271070	30.1	100.3
3	30	1268801	1273139	29.8	99.3