A Novel Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Silodosin and Tadalafil in its Synthetic Mixture
Avani N. Doshi1, Vidhi Kotadiya2*, Patel Amita3
1Professor, K. B. Raval College of Pharmacy, Kasturinagar, Shertha, Gandhinagar, Gujarat.
2Associate Professor, K. B. Raval College of Pharmacy, Kasturinagar, Shertha, Gandhinagar, Gujarat.
3PG Scholar, K. B. Raval College of Pharmacy, Kasturinagar, Shertha, Gandhinagar, Gujarat.
*Corresponding Author E-mail: avanidoshi20211@gmail.com
ABSTRACT:
The objective of this research is to design and validate a reliable Stability-Indicating RP-HPLC method for the simultaneous determination of Silodosin and Tadalafil in a synthetic mixture. A RP-HPLC method was established using an Agilent Eclipse XDB C18 column (150 mm × 4.5 mm ID × 5 µm) as the stationary phase. The mobile phase consisted of 0.1% TEA and ACN in a 60:40 (v/v) ratio. Sample injection was conducted at a flow rate of 1.0 mL/min, with detection set at a wavelength of 278 nm. Linearity within the concentration ranges of 40–120 µg/mL for Silodosin and 25–75 µg/mL for Tadalafil, with correlation coefficients (r˛) of 0.9997 and 0.9996, respectively. Precision studies confirmed method repeatability, as the percentage relative standard deviation (% RSD) remained below 2%. Robustness assessment also demonstrated % RSD values under 2%, indicating the method's reliability despite minor variations in analytical conditions. Accuracy was validated through a recovery study, confirming the method's suitability for precise quantitative analysis. Validation was performed in accordance with the International Conference on Harmonization (ICH) guidelines, demonstrating excellent precision, accuracy, linearity, specificity, sensitivity, and robustness.
KEYWORDS: RP- HPLC, Method Validation, ICH.
INTRODUCTION:
Benign prostatic hyperplasia (BPH) is a condition characterized by the non-cancerous enlargement of the prostate gland due to excessive cellular growth in the epithelial and fibromuscular tissues of the transition zone (TZ) and periurethral area. It is one of the most prevalent conditions among men, particularly as they age.
BPH is a key contributor to lower urinary tract symptoms (LUTS), which can significantly impact a patient’s quality of life. This age-related condition is especially common, affecting approximately 70% of men aged 70 and older1,2. Overactive bladder syndrome (OAB) is characterized by a sudden, strong urge to urinate, often accompanied by increased frequency and nocturia. These symptoms occur without an underlying condition that could otherwise explain them. It is crucial to rule out possible causes such as urinary tract infections or pelvic malignancies to ensure an accurate diagnosis. Silodosin and Tadalafil both the drugs are used to treat this condition.3 Drug Profile of both the drugs are given in Table 1.
The literature reveals that various methods has been developed for Silodosin and Tadalafil by UV, HPLC and HPTLC in single drug and combination with other drugs.
Table 1: Drug Profile of Silodosin and Tadalafil14-40
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Chemical name |
Silodosin |
Tadalafil |
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Structure |
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IUPAC name |
1-(3-hydroxypropyl)-5-[(2R)-2-[2-[2-(2,2,2 trifluoroethoxy)phenoxy]ethylamino]propyl]-2,3- dihydroindole-7-carboxamide |
(6R,12aR)-6-(1,3-Benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione |
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CAS No. |
1426173-86-5 |
171596-29-5 |
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Mol. weight |
495.5 g/mol |
389.4 g/mol |
|
Mol.formula |
C25H32F3N3O4 |
C22H19N3O4 |
No Analytical method was found for both the drug in single combination. So aim of Present work was to develop Stability indicating RP-HPLC method for the determination of Silodosin and Tadalafil, To conduct stress degradation study by ICH QIA (R2) and to perform Validation of method in accordance with ICH Q2 (R1) and ICH Q2 (R2) guidelines.
MATERIALS AND METHODS:
Silodosin and Tadalafil API were received as gift a sample. Chem pvt Ltd, Ahmedabad, Gujarat.All the reagents and Chemicals were of HPLC grade.
Instruments:
The Chromatographic technique was performed on Shimadzu (Model: LC 2010 CHT), Software: LC Solution. UV estimation was performed on Shimadzu UV-1800 Sartorius Analytical Balance with Accuracy: 0.001g, Lab India Digital pH Meter, Lab Junction Melting Point Apparatus, Agilent Cary 630 FTIR Spectrometer (Software: MicroLab Expert) were used for Method development.
Chromatographic conditions:
The chromatographic separation was achieved by using an Agilent Zorbax ODS (150mm x 4.6mm x 5µ) column using 0.1%TEA: ACN (60:40%v/v) as mobile phase. The flow rate was 1ml/min, and 278nm was used to analyze both the drugs. The injection volume was 20μl at ambient temperature.
This study focused on the development and validation of a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Silodosin and Tadalafil in a synthetic mixture. During method development, key chromatographic parameters were systematically optimized to enhance the efficiency of the HPLC system. These parameters included: Mobile phase composition, Flow rate, Detection wavelength, Analytical column and Column temperature. Various HPLC columns were evaluated, and the Agilent Eclipse XDB C18 (150mm × 4.5mm ID × 5µm) column was selected based on system suitability parameters, including: Retention time, Theoretical plates, Tailing factor, and Resolution.
Different mobile phase combinations of methanol, acetonitrile, and buffer in varying proportions were tested to achieve optimal separation and peak resolution.
A forced degradation study was conducted for Silodosin (SILO) and Tadalafil (TADA) to ensure that any impurities generated during stress conditions do not interfere with the main peaks or retention times in chromatographic analysis. Both the active pharmaceutical ingredients (APIs) and formulated samples were subjected to various stress conditions, including: Acidic hydrolysis, Basic hydrolysis, Oxidative degradation (hydrogen peroxide exposure), Photodegradation (UV light exposure). Chromatographic responses were recorded for each stress condition, and the percentage degradation was determined to assess the stability of the compounds under different conditions.
FORCED DEGRADATION CONDITION:
Acid Degradation:
Mixed 1 mL standard solution and 1mL 1 N HCl in a 10ml volumetric flask. Kept for 4 hours. Neutralized with 1 mL 1N NaOH. Volume made with diluent and injected into the HPLC.
Mixed standard solution 1ml + 1mL 1N NaOH in a 10 ml volumetric flask. Kept for 4 hours. Neutralized with 1 mL 0.1N HCL. Volume made with diluent and injected into the HPLC.
Mixed 1 mL standard solution and 1mL 3% H2O2 in 10ml volumetric flask. Kept for 12 hours.
Sample and standard solutions were kept in sunlight for 48 hours. Volume was made with diluent. Then injected into the HPLC.
Thermal degradation was performed by keeping API and samples in hot air oven at 60 °C 72 hours.
RESULTS:
Figure 1: Overlay UV spectra of SILO and TADA (Standard) Figure 2: Optimized chromatogram of SILO and TADA (Standard)
Figure 3: Overlaying linearity chromatogram of Silodosin and Tadalafil
Table 2 System suitability study
|
Sr. No |
System Suitability Parameters |
SILO |
TADA |
||
|
Mean (n = 6) |
% RSD |
Mean (n = 6) |
% RSD |
||
|
1 |
Retention Time |
3.06 |
1.56 |
7.45 |
0.45 |
|
2 |
Tailing Factor |
1.43 |
0.19 |
1.18 |
0.87 |
|
3 |
Theoretical Plate |
4149 |
0.13 |
6046 |
0.90 |
|
4 |
Resolution |
0.0 |
0.23 |
15.0 |
0.56 |
Table 3: % Degradation for Standard and Sample for TADA
|
Initial Standard Area |
1937319 |
Initial Sample Area |
2042456 |
||
|
Degradatio condition |
Area after degradation |
% Degradati on |
Degradation condition |
Area after degradation |
% Degradati on |
|
Acid |
1608847 |
17.0 |
Acid |
1665567 |
18.5 |
|
Base |
1805375 |
6.8 |
Base |
1815967 |
11.1 |
|
Peroxide |
1647686 |
15.0 |
Peroxide |
1666354 |
18.4 |
|
Thermal |
1904523 |
1.7 |
Thermal |
2004534 |
1.9 |
|
Photo |
1865969 |
3.7 |
Photo |
1998436 |
2.2 |
Table 4: Summary of RP-HPLC Validation Parameters of SILO and TADA
|
Sr. No. |
Parameter |
SILO |
TADA |
|
1 |
Accuracy (%Recovery) |
98.1 -100.7% |
99.8 -101.3% |
|
2 |
Regression equation |
y = 19670x + 9842.4 |
y = 36017x + 124015 |
|
3 |
Correlation co-efficient (r2) |
0.9997 |
0.9986 |
|
4 |
Limit of Detection (LOD) |
0.26 µg/ml |
0.04 µg/ml |
|
5 |
Precision (% RSD) |
||
|
Repeatability |
0.06 |
0.61 |
|
|
Intraday Precision |
1.73 |
0.74 |
|
|
Interday Precision |
0.25 – 1.76 |
0.54 - 1.67 |
|
|
6 |
Linearity and Range |
40 -120µg/ml |
25 -75 µg/ml |
|
7 |
Limit of Quantitation (LOQ) |
0.80 µg/ml |
0.13 µg/ml |
|
8 |
Specificity |
Specific |
Specific |
|
9 |
Robustness (% RSD) |
< 2 % in each parameters |
|
|
10 |
% Assay |
7.85, 98.7 ± 0.0786 |
5.04, 100.4± 0.054 |
CONCLUSION:
The present method was observed to be simple, specific, linear, precise, reliable, robust, and economical, and validated according to the regulations of ICH Q2B. From the stability studies of the present analytical method, the degradation behavior of was known, and it has been demonstrated that this method can successfully distinguish the degradation peak from the analytical peak. Symmetrical peaks have been observed within lesser analysis time, and the percent RSD values for all parameters were within the limits for the developed method. This shows that the results and assay achieved by this approach are in good agreement. Hence, the present analytical method developed can be used for the estimation of Silodosin and Tadalafil in a synthetic mixture, as well as for regular analysis in quality control purposes.
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Received on 28.07.2025 Revised on 15.09.2025 Accepted on 24.10.2025 Published on 02.01.2026 Available online from January 05, 2026 Asian J. Res. Pharm. Sci. 2026; 16(1):20-24. DOI: 10.52711/2231-5659.2026.00004 ©Asian Pharma Press All Right Reserved
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