An effective and sensitive stability-indicating chromatographic approach based on HPLC for silodosin assay
© The Author(s). 2016
Received: 3 April 2016
Accepted: 2 August 2016
Published: 17 August 2016
A stability-indicating reversed-phase high-performance liquid chromatography (HPLC) method with a high sensitivity was developed for the determination of silodosin (SIL) in the presence of hydrochlorothiazide (HCT) as an internal standard.
Chromatographic separation of SIL and IS were successfully achieved on an Agilent ZORBAX CN column with an isocratic mobile phase composed of a mixture of methanol:acetonitrile:ammonium acetate (pH 4.0; 0.015 M) (40:30:30, v/v/v) at a flow rate of 1.3 mL min−1. The drugs were quantified using a photodiode array detector set at a wavelength of 270 nm. The reversed-phase HPLC method has been validated as per International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines to determine SIL in pharmaceutical dosage form.
The proposed method showed a good linearity in the concentration range of 4.0–600.0 μM with a lower detection limit of 85.0 nM under optimized conditions. The statistical performance of the fully validated HPLC method was compared to our developed sensitive spectrofluorimetric method, and the performance results of the proposed HPLC method were considerably satisfactory. The validated method was successfully applied to quantify the SIL in capsules, and the corresponding recovery value was found to be 99.5 %.
The validated HPLC method may be a promising alternative analytical tool for routine analysis of SIL in pharmaceutical samples.
The literature revealed that various analytical methods have been reported to determine the SIL in pharmaceutical or clinical samples including UV spectrophotometry (Jahan and Malipatil 2014b), spectrofluorimetry (Bhamre and Rajput 2014), high-performance liquid chromatography (HPLC) (Jahan and Malipatil 2014a ; Aneesh and Rajasekaran 2012; Vali et al. 2012), high-performance thin-layer chromatography (Sayana et al. 2012), ultra high-performance liquid chromatography (UHPLC) (Shaik et al. 2014; Prasad et al. 2012) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Zhao et al. 2009) and electrochemicalsensing (Er et al. 2015) methods. Recently, the authors have attached great importance develop the sensitive and reliable analytical methods for determination of biological and drug molecules at nano-molar levels. In this point, we thought that an effective and highly sensitive analytical tool such as HPLC is required with a short response time for SIL assay. In addition, the spectrophotometric approach is also preferred especially in the detection of drug molecules due to its many advantages such as cost effectiveness, easy operation, high sensitivity and repeatability, rapid response time and low detection limit (Tekkeli and Önal 2011; Walash et al. 2013). The present study has indicated accurate and efficient analytical methods based on reversed-phase HPLC and spectrofluorimetry for the determination of SIL in a pharmaceutical sample.
Chemicals and reagents
SIL powder was gifted from Recordati Pharm. Company. Urorec® (containing 8.0 mg SIL per capsule) capsules were purchased from a local market in Ankara, Turkey. The internal standard (IS) was used as a hydrochlorothiazide (HCT) in HPLC measurements. The methanol and acetonitrile solutions for HPLC studies were purchased from J.T. Baker (Phillipsburg, NJ) with HPLC grade. The other chemicals and reagents were purchased from Sigma-Aldrich Company (Germany) and were of analytical grade. All solutions and mobile phase were prepared with ultrapure water using Waters Milli-Q Plus purification system.
Instrumentation and conditions
HPLC analyses were performed by an Agilent 1100 series LC system (Agilent Technologies, Wilmington, USA) equipped with an Agilent series G-1315B diode-array detector (DAD), G-1313A ALS autosampler, G1311A Quat pump and G1379A degasser. Chromatographic separation was performed on a Agilent ZORBAX CN column (reversed-phase) (150 mm × 4.6 mm, 5 μM) in isocratic mode. Data were collected and processed by the use of Agilent ChemStation. The mobile phase consisted of a mixture of aqueous 0.015 M ammonium acetate (pH 4.0):methanol:acetonitrile in the ratio (30:40:30, v/v/v). The pH of the ammonium acetate was adjusted to 4.0 by addition of NaOH and HCl, and the mixture was pumped at 25 °C with a flow rate of 1.3 mL min−1. The detection was achieved at 270 nm, and the injection volume was 10 μL. The mobilephase mixture was filtered through a 0.45μm membrane filter (Millipore, Bedford, MA) and degassed under ultrasonic bath before HPLC analysis. The quantification of SIL was based on peak area ratio using IS.
Fluorescence spectra were measured by Agilent Cary Eclipse spectrofluorometer (CA, USA) equipped with a Xenon flash lamp. The slit widths for excitation and emission monochromators were fixed at 10 nm. All measurements were performed in a 1.0cm quartz cell at room temperature (25 °C).
Preparation of standard solutions
Fifty milligrams each of standard SIL and HCT powder were accurately weighed and dissolved in a 100-mL mixture of methanol:water (1:1, v/v) by sonication for 10 min. The SIL standard solutions were diluted by the mixture of methanol:water (1:1, v/v) and phosphate buffer (pH 6.0) to obtain the required workingrange concentrations for HPLC and spectrofluorimetry, respectively. Each of the SIL standard solution contains 25 ppm HCT solution during HPLC analysis. The solutions were filtered through a 0.45μm membrane filter before injection into the HPLC system.
Preparation of Urorec® capsules
For both HPLC and spectrofluorimetry, ten Urorec® capsules were carefully weighed and powdered to get a homogenous fine powder in a mortar. An appropriate weight of this powder equivalent to one capsule content was weighed, transferred into the calibrated flask and then dissolved in the mixture of methanol:water (1:1, v/v) in an ultrasonic bath. The final mixture was filtered by the use of a 0.45μm membrane filter, and the filtrate was diluted with the mixture of methanol:water (1:1, v/v) and phosphate buffer solution (pH 6.0) to obtain the certain concentration in the linearity range of SIL for HPLC and spectrofluorimetry, respectively.
Forced degradation and stability-indicating tests
Ten milligrams each of standard SIL and HCT were dissolved to prepare the stock solution in a 25mL mixture of methanol:water (1:1, v/v) by sonication for 10 min. After forced degradation process, each solution was filtered through a 0.20μm PTFE syringe filter before injection into the HPLC system
In the presence of IS, 2.5 mL of 1.0 M HCl was added to 7.5 mL stock solution, and the mixture was kept at 80 °C for 1 h under reflux, cooled and neutralized with 1.0 M HCl to pH 7.0. Then, 6.25 mL of the solution was made up to 25 mL with ultrapure water. Finally, the solution was filtered through a 0.20μm PTFE syringe filter.
In the presence of IS, 2.5 mL of 1.0 M NaOH was added to 7.5 mL stock solution, and the mixture was kept at 80 °C for 1 h under reflux, cooled and neutralized with 1.0 M NaOH to pH 7.0. Then, 6.25 mL of the solution was made up to 25 mL with ultrapure water. Finally, the solution was filtered through 0.20-μm PTFE syringe filter.
In the presence of IS, 2.5 mL of 5 % H2O2 was added to 7.5 mL stock solution, and the mixture was kept at 80 °C for 1 h under reflux then cooled, and the volume of the mixture was made up to 25 mL with ultrapure water. Finally, the solution was filtered through 0.20-μm PTFE syringe filter.
Ten milligrams each of SIL and HCT powders were kept at 80 °C for 24 h. After that, each powder was dissolved in a mixture of methanol:water (1:1, v/v). An aliquot of these solutions was dissolved to get a required solution consisting of 50 ppm SIL and 25 ppm HCT. The solution was filtered through 0.20-μm PTFE syringe filter.
Results and discussion
Optimization of HPLC conditions
Firstly, HPLC conditions were optimized to obtain a desired peak with high purity and resolution. Therefore, the various parameters affecting the peak shape, retention time and resolution of SIL were investigated in detail. The separation efficiency of Agilent ZORBAX CN column (150 mm × 4.6 mm, 5 μm) was compared to the monolithic column (Supelco® C18; 150 mm × 4.6 mm, 5 μm) for the determination of SIL under the same conditions, and the proposed column was chosen for the further optimization parameters.
During our preliminary experiments, the series of aqueous mobile phases containing buffer solutions with the different pH values in combination with different organic modifiers including the different ratios of acetonitrile, methanol and ammonium acetate were tested for obtaining the optimum separation conditions. Acetonitrile, methanol and ammonium acetate were selected as the eluents. The chromatographic analysis time of SIL was shortened with high organic solvent content, and also, the buffer solutions in the mobile phase ensured stable chromatographic retention times preventing broad peaks.
The effect of the mobilephase pH on the retention time and peak shape of the analyte was studied especially in the acidic region. The best retention time and peak shape of SIL was achieved at pH 4.0 acetate buffer. The best separation was achieved with the mobile phase consisting of methanol:acetonitrile:acetate buffer (pH 4.0) (40:30:30, v/v/v). HCT was chosen as the IS as its retention time did not prolong the analysis time and indicated no interference effect on the chromatographic peak of SIL. The calibration curves of SIL for HPLC analysis were constructed by plotting the peak area ratio of drug molecule to IS against the concentration of the drug.
Optimization of spectrofluorimetry conditions
Effect of pH and excitation wavelength on fluorescence intensity
It is a known fact that the intensities of fluorescent molecules are connected with the pH value of the medium and excitation wavelength. The influence of pH on the fluorescence intensity of SIL was investigated using different pH values in the range from 2.0 to 10.0 with the three replicate measurements. The maximum fluorescence intensity of SIL was observed at pH 6.0 as shown in Fig. 1a. The effect of the excitation wavelength on fluorescence intensity was also performed with three replicate measurements in the range of 310–360 nm. It was found that maximum fluorescence intensity of SIL was obtained at excitation wavelength of 330 nm in pH 6.0 phosphate buffer as shown in Fig. 1b.
Chromatographic and spectrofluorimetric approaches have been useful techniques for the determination of drug or biologically important molecules in real samples for many years, and these techniques offer a simple way to quantify the drug molecules especially in pharmaceutical formulations (Belal et al. 2013; Antunes et al. 2013). Therefore, the presented study aims to develop validated analytical methods for the determination of SIL in pharmaceutical samples.
System performance parameters obtained by HPLC for SIL
Reference value (Center for Drug Evaluation and Research (CDER) 1994)
Retention time, t r
Capacity factor, K′
K′ > 2.0
Resolution, R s
R s > 2.0
α > 1.0
Theoretical plates, N (plates/column)
N > 2000
Tailing factor, T
T ≤ 2.0
Validation results obtained from HPLC for the quantitative determination of SIL
Linearity range (μM)a
Correlation coefficient (R 2)
Repeatability of peak area, (RSD %)c
Repeatability of peak area, (RSD %)d
The limit of detection (LOD) for SIL was found to be 7.7 nM calculated from related equation (S/N = 3). The similar study claimed that a narrow working range such as 0.1–3.0 μg mL−1 for SIL are obtained at the excitation wavelength of 340 nm, and the best results for SIL were obtained at the excitation wavelength of 272 nm (Bhamre and Rajput 2014). Contrary to these results, the fluorescence intensity of SIL was gradually decreased with decreasing excitation wavelength from 330 to the less excitation wavelength in our study as shown in Fig. 1b. In this point, we presented a highly large linear range for SIL at the excitation wavelength of 330 nm in the buffer solution system.
Validation results obtained from spectrofluorimetry for the quantitative determination of SIL
λex = 330, λem = 460
Linearity rangea (μM)
Correlation coefficient (R 2)
Repeatability of peak intensity (RSD %)c
Repeatability of peak intensity (RSD %)d
The comparison of developed analytical methods used in determination of SIL
Linearity range (μM)
(Jahan and Malipatil 2014b)
(Bhamre and Rajput 2014)
(Jahan and Malipatil 2014a)
(Aneesh and Rajasekaran 2012)
(Vali et al. 2012)
(Shaik et al. 2014)
(Zhao et al. 2009)
(Er et al. 2015)
Silodosin assay in Urorec® capsules
Analysis of SIL in capsules using proposed methods
Average recovery (%)
RSD of recovery (%)
Forced degradation behaviour
Results of forced degradation study of SIL drug using the developed stability-indicating HPLC method
Heat temperature (°C)
Acidic (1.0 M HCl)
Alkali (1.0 M NaOH)
Oxidative degradation (5 % H2O2)
A highly sensitive and effective validated reversed-phase HPLC method was successfully developed with a low LOD value for SIL assay. The SIL in the presence of HCT were subjected to forced degradation under several stress conditions. The satisfactory results were achieved from degradation studies, which revealed that the method was stability indicating. Besides, the spectrofluorimetric approach has drawn attention with satisfactory results in the detection of SIL in a pharmaceutical sample. The proposed methods have significant advantages such as rapid, sensitive, not needing a fluorescent agent and derivatization reactions, good reproducibility and cost effectiveness. The results showed that the developed analytical methods could be a very promising alternative for the determination of SIL in routine drug and clinical application.
This research has been supported by the Ankara University Scientific Research Projects Coordination Unit (Project Number: 15B0237001, 2015). The authors thank the Recordati Company for supplying the drug.
Both authors equally contributed in experimental design, analysis results, writing and proofing and approved the final manuscript. Experimental work was performed by EE.
The authors declare that they have no competing interests.
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