A Novel Analytical Method for Simultaneous Quantification of Silodosin and Tadalafil by RP- HPLC

The Reverse phase HPLC method was developed for simultaneous determination of Silodosin and Tadalafil in single analytical method. Chromatographic separation was achieved on a Supelco C8 (150mmx4.6mm, 5μm) column applying an isocratic elution based on premix of potassium phosphate dibasic buffer pH (4.3) and acetonitrile in the ratio of (70:30 v/v) as mobile. Validation parameters specificity, precision and robustness have been observed to be desirable over the concentration ranges of 80-240 μg/ml for Silodosin and 50-150 μg/ml for Tadalafil in accuracy parameter and 128-192 μg/ml for Silodosin and 80-120 μg/ml for Tadalafil in linearity parameter. All the variables have been studied to optimize the chromatographic conditions. The optimized approach verified through validation and confirmed to be intended purpose for the quality control of the mentioned drugs, as per ICH guidelines. For simultaneous quantification of Silodosin and Tadalafil, the developed method was found to be genuinely exact precise, accurate, linear, fast and cost effective.


INTRODUCTION
In the pharmaceutical industry, all manufactured products need to be of the highest quality to ensure the least risk to patients. To guarantee that goods pass certain standards, researchers, manufacturers and developers use various technical equipment and analytical techniques, including liquid chromatography, during the development process [1]. It's possible that the medicine or drug combination isn't included in any pharmacopoeias. Due to patents, legislation, and other factors, a proper analysis process for the medicine may not be available in the literature. Due to the interference caused by the formulation excipients, analytical techniques for the drugs in the form of a formulation may not be available. Analytical methods for measuring drug concentrations in biological fluids may not be available [2].
Many multi-component medicines have been developed in the modern pharmaceutical business, and we can now quantify them with greater accuracy [3]. In quality control, liquid chromatography (LC) is the most extensively used analytical technique for determining the identification and amount of analytes and impurities in production batches. The pharmaceutical sector is heavily regulated. It is being scrutinized more closely by government regulatory agencies and public interest groups in order to reduce costs and ensure the timely delivery of safe and effective products to market. Both industry and regulatory agencies have turned their attention to the quality of drug products. Increased pressure on pharmaceutical analysts to supply accurate and precise analytical data in the quickest feasible time has resulted from faster drug research and drug product development processes, as well as increased requirements from industry and regulatory bodies [4] A detailed literature review shown that individual analytical HPLC method is available for the determination of Silodosin and Tadalafil [5,6,7]. However few analytical methods are available for simultaneous determination of Silodosin in combination with other drugs except with Tadalafil [8]. In the same way few analytical methods are available for simultaneous determination of Tadalafil in combination with other drugs except with Silodosin [9,10]. But so far there is no single analytical HPLC method is available for simultaneous estimation of Silodosin and Tadalafil. The goal of this study is to establish a simple, reproducible, linear, precise single analytical method for simultaneous quantification of Silodosin and Tadalafil.

Silodosin
Silodosin is a kind of silodosin that (Brand Name Rapaflo) Silodosin is a drug used to treat the symptoms of benign prostatic hyperplasia. It has a strong uroselectivity and acts as a 1adrenoreceptor antagonist (selectivity for the prostate) [11]. The great selectivity, on the other hand, appears to be the reason of silodosin's common side effect of loss of seminal emission. Silodosin is a prescription drug that is used to treat a variety of conditions [12]. Silodosin is a novel selective 1-adrenoreceptor antagonist with a high pharmacologic selectivity in the alphablocker class [13].

Tadalafil
Tadalafil is used to treat male penile erectile dysfuctions (ED) by inhibiting the enzyme phosphodiesterase, which reduces the metabolism of cyclic guonosine monophosphate (cGMP), causing smooth muscle relaxation in the corpus cavereosus, with an onset time of 30-45 minutes [14].

METHODS AND MATERIALS
The liquid chromatography consisted of a Shimadzu HPLC system model LC 2010 High-Performance Liquid Chromatography. For the RP-HPLC system, a Supelco C8 (150 mm x 4.6 mm, 5 µm) column was used. A Photodiode array detector (PDA) with an automated sample injector integrated with the system. Empower software was used to monitor and integrate the output signal. The hydrogen ion concentration (pH) of the buffer solutions was adjusted and determined using a digital pH meter. Active Pharmaceutical ingredient (API) of Silodosin and Tadalafil were supplied as gift from Metrochem API Pvt Ltd, Hyderabad. HPLC grade Methanol, Acetonitrile, Triethylamine, Miili-Q water, Ortho-Phosphoric acid (OPA) and Potassium Phosphate dibasic of analytical grade were obtained from Finar Chemicals Ltd.

Mobile Phase and Chromatographic Conditions
Potassium phosphate dibasic buffer solution was prepared by dissolving 3.48 gm of potassium phosphate dibasic in 1000 ml of HPLC grade water. Add accurately 2 mL of triethylamine in this solution and mix. The solution pH was adjusted to 4.3 with orthophosphoric acid. Mix buffer solution pH 4.3 and acetonitrile in the ratio of 70:30 v/v and use as a mobile phase. Diluent used mixture of milli-Q water and acetonitrile in the ratio of 50:50 v/v.

Standard Preparation
Standard solutions of Silodosin and Tadalafil were prepared by dissolving about 16 mg of silodosin working standard and 10 mg of Tadalafil working standard into a 100 mL volumetric flask, add about 70 mL of diluent, sonicate to dissolve and make up to volume to100 mL with diluent.

Sample Preparation
The average weight was derived after accurately weighing of 10 tablets. The tablets were crushed and powdered and a quantity of powder equivalent to 16 mg of Silodosin and 10 mg of Tadalafil was transferred into 100 mL volumetric flask and diluent was added about 70 ml. The solution was sonicate for 20 min. It was diluted to volume with diluent and mix well. A portion of the solution was filter with the 0.45 µm membrane filter.

Chromatographic Conditions
Shimadzu, Model: LC 2010, Photodiode array detector (PDA), with an auto sampler injector. The output signal was monitored and integrated using Empower software. The separation was successfully achieved on Supelco C8 (150x4.6mm, 5µm) column. The column temperature was maintained at 30°C and the eluent was monitored at 232 nm using a PDA detector. The injection volume was 10 μl. Mixture of phosphate buffer (pH 4.3) and acetonitrile in the proportion of 70:30 v/v as flow rate of 1.0 mL/min was used as a mobile phase with isocratic method.

Method Validation
The parameters for method validation were carried out in accordance with ICH recommendations [19].

Specificity
The specificity was established by injecting blank, placebo, sample, spiked sample and individual impurities into the system. All samples solutions were prepared as per developed method.
For analyte identification, chromatograms were recorded and retention times from sample and standard preparations were compared. No interference was observed from the blank, placebo & known impurities at the retention time of Silodosin and Tadalafil peak. As purity angle was found less than purity threshold for both drugs (Table-1). Blank, Standard solution and sample solution chromatograms are shown in Fig. 1, Fig. 2 & Fig. 3.

Precision
The system precision was verified by injecting six replicate injections of standard solutions. Calculate the mean assay and percent relative standard deviation (%RSD) of area counts of Silodosin and Tadalafil peak ( Table 2). The method precision was verified by injecting six replicate injections of sample solution. Calculate the mean assay and percent relative standard deviation (%RSD) of area counts of Silodosin and Tadalafil peak ( Table 2). The intermediate precision was verified by injecting six replicate injections of sample solution on a different day by a different analyst and analyse on a different instrument using the column of different serial number. Calculate the mean assay and percent relative standard deviation (%RSD) of area counts of Silodosin and Tadalafil peak (Table 3).

Linearity
In the linearity parameter, concentration of Silodosin and Tadalafil response were determined in the range of 80%-120% of standard solution. Calibration curves were plotted between analyte concentration and peak response. MS-Excel was used to determine the slope, intercept, and correlation coefficient. The calibration data of Silodosin and Tadalafil is given in Table 4, while Fig. 4 & Fig. 5 represents calibration curve of both drugs respectively.

Accuracy
The accuracy was determined by adding known quantities of the analyte to the placebo. A 3-fold measurement at 50% (Level 1), 100% (Level 2), and 150% (Level 3) of sample concentration respectively (9 Determinations in total) was carried out. Data for Silodosin and Tadalafil are shown in Tables 5-6.

Robustness
Robustness of the method shall be demonstrated by deliberately changing the chromatographic parameters and monitoring system suitability parameters under each condition. Prepare standard solutions as described in method to be injected under each of the variable conditions such as wavelength of detection by ± 3 nm, flow rate by ± 10%, pH of buffer by ±0.2 unit and column oven temperature by ± 5°C.

Specificity
There was no interference of blank and placebo at the retention time of Silodosin and Tadalafil peak. Also purity angle was found less than from purity threshold for both drugs. Hence the method was found to be specific.

Linearity and Range
The correlation coefficients for Silodosin and Tadalafil were found to be 0.9991 and 0.9992 respectively between 80%-120% range of the target concentration of analyte.

Precision
The % RSD was found 0.56 and 0.54 for system precision and 0.29 and 0.44 was found for repeatability study for Silodosin and Tadalafil respectively.

Robustness
All system suitability criteria were found within acceptance limit during small but deliberately changes in chromatographic conditions that indicates developed method is robust.

Stability of Analytical Solution
Prepare the standard and sample solution as per developed method, keep the solutions at 25°C. Inject at different time intervals. Solution stability of standard and sample solution was found for 24 hours.

CONCLUSION
Above results concluded that developed method is specific, precise, linear, reproducible and rugged. This method is validated according with ICH guideline. During analytical method validation, results were found satisfactory. Simultaneously quantification of Silodosin and Tadalafil in single analytical method with shorter run time shows this method cost-effective, time saving and can be used for routine analysis in industries.

DISCLAIMER
There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.