Development of Stability-Indicating UHPLC Method for the Quantitative Determination of Silodosin and Its Related Substances

Journal of Chromatographic Science 2014;52:646– 653 doi:10.1093/chromsci/bmt094 Advance Access publication July 11, 2013 Article Development of Stability-Indicating UHPLC Method for the Quantitative Determination of Silodosin and Its Related Substances Jafer Vali Shaik1,2*, Shantikumar Saladi4 and Shakil S. Sait3 1 United States Pharmacopeia—India Private Limited, Research and Development Laboratory, ICICI Knowledge Park, Hyderabad, India, Department of Chemistry, Jawaharlal Nehru Technological University, Hyderabad, India, 3Dr. Reddy’s Laboratories, Ltd., Hyderabad, India, and 4National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India 2 *Author to whom correspondence should be addressed. Email: Received 17 December 2012; revised 25 May 2013 A novel, specific and stability-indicating reversed-phase (RP) ultrahigh-performance liquid chromatography (UHPLC) method, which is mass compatible, was developed and validated for the quantitative determination of silodosin and its related substances. Silodosin was subjected to stress conditions like hydrolysis (acid and basic), oxidation, photolysis and thermal degradation, as per the guidelines of the International Conference Harmonization, to show that the method is stability-indicating. The proposed UHPLC method has a resolution of greater than 2.0 between silodosin and its process-related impurities. The chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column (50 3 4.6mm i.d.; particle size, 2.7 mm). The method employed a linear gradient elution using a mobile phase consisting of acetonitrile and 10 mM ammonium acetate buffer with 0.1% triethyl amine, with pH adjusted to 6.0, monitored at 273 nm. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The known process impurities were separated and their structure was confirmed by using liquid chromatography –mass spectrometry and direct mass analysis. in bulk and pharmaceutical dosage forms. These include ultraviolet (UV) (8), high-performance liquid chromatography (HPLC) (9) and liquid chromatography–mass spectrometry (LC–MS) (10, 11) methods, but no study has reported a stability-indicating ultra-high-performance liquid chromatography (UHPLC) method for the quantification of the process related impurities and degradants of silodosin. The objective of the present study was to develop a stability-indicating reversed-phase (RP) UHPLC method for the quantitative determination of process impurities and for the separation and confirmation of possible major degradants related to silodosin by using LC–MS. The optimized UHPLC method was able to separate silodosin, its three known process impurities and the degradants that were formed during degradation with good resolution. In the present study, silodosin, two key starting materials were used: Impurity 1 (Figure 1B) and Impurity 2 (Figure 1C); Impurity 3 (Figure 1D), which is an intermediate formed during the synthesis of silodosin, was also used. Experimental Introduction Silodosin (Figure 1A), chemically 1-(3-hydroxypropyl)-5[(2R)-2-(f2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylgamino)propyl]-2,3-dihydro-1H-indole-7-carboxamide, is a medication for the symptomatic treatment of benign prostatic hyperplasia (1). It acts as an a1-adrenoceptor antagonist with high uroselectivity (2). It is used by men to treat the symptoms of an enlarged prostate (benign prostatic hyperplasia; BPH), which include difficulty in urinating (hesitation, dribbling, weak stream and incomplete bladder emptying), urinary frequency and urgency. Silodosin is approved for the treatment of the signs and symptoms of BPH in Europe, the United States, and Japan. The trade names of silodosin are Rapaflo (US), Silodyx (Europe), Rapilif (India), Silodal (India) and Urief (Japan). Silodosin received its first marketing approval in Japan in May of 2006 under the trade name Urief, which is jointly marketed by Kissei Pharmaceutical Co. and Daiichi Sankyo Pharmaceutical Co. Silodosin has one chiral center and is used as a single enantiomer (R) (3). BPH is one of the most common diseases in men, with an increasing prevalence rate with age (4, 5). BPH is a histological diagnosis characterized by the proliferation of smooth muscle and epithelial cells within the prostatic transition zone (6, 7). Literature reveals that there are only few analytical methods for estimation of silodosin Chemicals and reagents Ammonium acetate (CH3COONH4), glacial acetic acid (99%) and triethyl amine were purchased from Merck (Mumbai, India). Silodosin active pharmaceutical ingredient (API) with more than 99% purity, two key starting materials and one intermediate that were used for the synthesis of Silodosin (Impurity 1, Impurity 2 and Impurity 3) were obtained as gift samples from MSN Labs (Hyderabad, India). Instrumentation An Agilent UHPLC 1260 Infinity series with diode array detector (DAD) was used. The system was equipped with a temperature control oven. Data acquisition and processing used Waters Empower software. A Thermo Scientific LC–MS was used to confirm the masses of process related impurities. The potency of silodosin API and its three process impurities was evaluated by using a thermogravimetric analysis (TGA) instrument (Model TA-Q50). Preparation of stock, standard and test solutions The stock solutions of the three impurities of silodosin were prepared by separately dissolving 10 mg of each impurity in 20 mL of diluent (100%). A series of dilutions were made by using # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: Figure 1. Sturctures of (A) Silodosin, (B) Impurity-1, (C) Impurity-2, (D) Impurity-3. 100% solutions of Impurity 1, Impurity 2 and Impurity 3. The sample solution was prepared by weighing approximately 10 mg of silodosin into a 20 mL volumetric flask; the drug was dissolved and diluted to 20 mL with diluent. System suitability test Ten milligrams of silodosin were dissolved in 20 mL of diluent and spiked with all three impurities at a level of 0.15%. As a part of system suitability tests, two criteria were defined: (i) resolution between silodosin and Impurity 1 should not be less than 2.0; (ii) tailing factor of silodosin should not be more than 1.5. Chromatographic conditions The chromatographic separation was conducted on an RP Agilent Poroshell 120EC-C18 column (50  4.6 mm i.d.; particle size, 2.7 mm). The temperature was maintained at 288C and the mobile phase consisted of 10 mM ammonium acetate buffer mixed with 0.1% triethyl amine, pH 6.0 adjusted with glacial acetic acid. The optimized flow rate and injection volume were 0.7 mL/min and 10 mL. The final concentration of the sample was 0.5 mg/mL and detection was conducted by using a photodiode array (PDA) detector at 273 nm. The diluent consisted of a mixture of acetonitrile and mobile phase A (1:1). The UHPLC gradient program was as follows (...truncated)