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

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1 Journal of Chromatographic Science 2014;52: doi: /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 Shaik 1,2 *, Shantikumar Saladi 4 and Shakil S. Sait 3 1 United States Pharmacopeia India Private Limited, Research and Development Laboratory, ICICI Knowledge Park, Hyderabad, India, 2 Department of Chemistry, Jawaharlal Nehru Technological University, Hyderabad, India, 3 Dr. Reddy s Laboratories, Ltd., Hyderabad, India, and 4 National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India *Author to whom correspondence should be addressed. shaikvali2008@gmail.com 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 ( mm 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. 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 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 Chemicals and reagents Ammonium acetate (CH 3 COONH 4 ), 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 journals.permissions@oup.com

2 Figure 1. Sturctures of (A) Silodosin, (B) Impurity-1, (C) Impurity-2, (D) Impurity % 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 ( 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: 0 min/10% solution B, 6 min/90% solution B, 8 min/90% solution B and 12 min/10% solution B. Potency evaluation Approximately 5 to 10 mg of the sample was analyzed with a 58C linear ramp to 1208C by using a TGA (TA-Q50 Model) instrument. Evaluation of relative retention factor Approximately six solutions containing a mixture of drug substance and impurities were prepared with respect to a specified limit of 0.15% (i.e., 0.15% was considered to be 100%, then 50, 75, 100, 125, 150 and 200%). The prepared solutions were injected into the UHPLC system. Method Validation The developed method was validated according to the guidelines of the International Conference on Harmonization (ICH) (12, 13) with respect to parameters like linearity, accuracy, solution stability, limit of detection (LOD), limit of quantitation (LOQ), precision, ruggedness and robustness. Specificity and forced degradation study Specificity represents the ability of an analytical method to measure the analyte freely from interference as a result of blank and degradation products. The specificity was evaluated by forced degradation studies, which ensured the selectivity of the proposed analytical method. Forced degradation studies were conducted as per ICH guidelines. Hydrolytic degradation was tested by preparing silodosin solutions (5 mg/ml) in 0.1 N HCL, 0.1 N NaOH and distilled water and refluxing at 908C for 7 h. For oxidative studies, silodosin solution (1.0 mg/ml) was prepared in 0.05% hydrogen peroxide and kept at room temperature for 10 min. During the development study, even in 1.0% hydrogen peroxide, silodosin was not stable; within the sample preparation and neutralization time, it completely degraded, but stable results were obtained with 0.05% hydrogen peroxide. During base, peroxide and neutral degradation, silodosin was dissolved Development of Stability-Indicating UHPLC Method for the Quantitative Determination of Silodosin and Its Related Substances 647

3 Figure 2. A typical chromatogram of Silodosin with impurities spiked (Poroshell 120 EC-C18 50mm 4.6mm i.d.; particle size, 2.7 mm) at a temperature of 288C and flow rate of 0.7mL/min). in approximately 5 to 10 ml of acetonitrile and the volume was completed with the respective diluent under each condition because of its insolubility in that particular diluent. Thermal degradation was conducted by exposing silodosin to 1058C in a hot air oven for two days. In photo degradation, the silodosin sample was exposed to an overall illumination of no less than 1.2 million lux h of visible light in a photostability chamber Similarly, the sample was exposed to UV radiation, providing an overall energy of no less than 200 W h/m 2 in a photostability chamber. All stressed samples were suitably diluted with the mobile phase (acidic and basic hydrolytic stressed samples were appropriately quenched) to produce concentrations of 0.5 mg/ml and injected into the chromatographic system. Sensitivity The sensitivity was determined by establishing the LOD and LOQ for silodosin, Impurity 1, Impurity 2 and Impurity 3. The LOD and LOQ were estimated by injecting a series of dilute solutions with known concentrations at signal-to-noise ratios of 3:1 and 10:1, respectively. Precision The precision of the related substance (RS) method was performed by injecting six individual preparations of silodosin (0.5 mg/ml) spiked with 0.15% (100%) each of Impurity 1, Impurity 2 and Impurity 3. The relative standard deviations (RSDs) were calculated for the area percentage of each impurity. Linearity and range A linearity test solution for the RS method was prepared by diluting the stock solutions of silodosin and its three impurities to Table I Potency and RRF Results Sample Name Potency (%) RRF values 01 Silodosin Impurity Impurity Impurity Table II Specificity Results* Sample Stress condition Degradation observed (%) Retention times of major degradants (min) 01 Acid hydrolysis , Base hydrolysis 0.27 No major degradants 03 Water hydrolysis 0.37 No major degradants 04 Oxidative degradation 05 Thermal , 5.14 degradation 06 Photo degradation 0.25 No major degradants 07 UV degradation 0.34 No major degradants *Note: Silodosin is highly sensitive to peroxide, because when the sample was dissolved in 0.05% peroxide and kept under room temperature for 10 min, approximately 15% degradation was observed. Silodosin is highly stable toward base, photo, UV and neutral degradation. Silodosin is slightly sensitive to acid and thermal conditions. the required concentrations. The solutions were prepared at seven levels (LOQ to 200%), with 0.15% considered to be 100% (i.e., LOQ, 50, 75, 100, 125, 150 and 200%), and subjected to linear regression analysis with the least-squares method. The calibration equation obtained from regression analysis was used to calculate the corresponding predicted responses. 648 Vali et al.

4 Figure 3. Chromatograms of (A) Peroxide degradation (10 min at RT with 0.05% H 2 O 2 ), (B) Acid degradation (refluxed at 908C for 7 hrs with 0.1N HCl), (C) Thermal degradation (1058C for 2 days) Accuracy The accuracy of the method was determined by analyzing silodosin (n ¼ 3) spiked with (50 to 150%) 0.075, 0.15 and 0.225% of the impurities. Ruggedness The ruggedness of the method was examined by performing a precision study, which consisted of injecting six individual preparations of 0.5 mg/ml of silodosin spiked with 100% of each Development of Stability-Indicating UHPLC Method for the Quantitative Determination of Silodosin and Its Related Substances 649

5 impurity using different columns, different analysts and different systems from the same laboratory. Robustness To determine the robustness of the developed method, experimental conditions were deliberately changed and the resolution (Rs) was evaluated between silodosin, Impurity 1, Impurity 2 and Impurity 3. The flow rate of the mobile phase was 0.7 ml/ min. To study the effect of flow rate on the developed method, +0.2 units of flow were changed (i.e., 0.5 and 0.9 ml/min). The effect of column temperature on the developed method was studied at 23 and 338C instead of 288C. The effect of ph on the resolution of impurities was studied by varying +0.2 ph units (i.e., buffer ph altered from 6.0 to 5.8 and 6.2). In all of the varied conditions, the components of the mobile phase were constant. System suitability and 100% spiked sample were injected under these conditions to check the resolution of three impurities. Solution stability Thestabilityofthesolutionwastestedforsampleandsystem suitability solutions by storing them for up to 48 h at room temperature. Results The gradient mobile phase employed in the present study included a 10 mm ammonium acetate buffer mixed with 0.1% triethyl amine, ph 6.0 adjusted with glacial acetic acid. Mobile phase A consisted of 90% buffer and 10% acetonitrile (90:10, v/v) and Table III Linearity Results Sample Name Range (mg/ml) Correlation coefficient (r) 01 Silodosin Impurity Impurity Impurity Table IV Accuracy Results Level of Accuracy Impurities Impurities added (mg/ml) Impurities recovered (mg/ml; n ¼ 3) 50% % % Impurities recovered (%; n ¼ 3) mobile phase B consisted of 95% acetonitrile with 5% Milli-Q water (95:5, v/v). The optimized flow rate was 0.7 ml/min, the column oven temperature was set at 288C and the elution was conducted up to 12 min. The HPLC gradient program was set as follows: 0 min/10% solution B, 6 min/90% solution B, 8 min/90% solution B and 12 min/10% solution B. The diluent was a mixture of acetonitrile and mobile phase A (1:1). A typical chromatogram in which separation was achieved is shown in Figure 2. The flow rate was 1.0 ml/min with UV detection at 274 nm. Method validation studies Specificity The results of forced degradation studies with approximate percentage of degradation and retention times of major degradation products are tabulated in Table II. The chromatograms are shown in Figure 3. Sensitivity The LOD of Impurity 1, Impurity 3 and silodosin was approximately mg/ml and the LOQs were between and mg/ml. The LOD of Impurity 2 was approximately mg/ml and the LOQ was approximately mg/ml. A precision study was also conducted at the LOQ level by injecting six individual preparations of Impurity 1, Impurity 2 and Impurity 3 and the RSDs were calculated for the peak areas of all impurities. The precision values at LOQ concentration for the impurities were below 2%. The recovery values at LOQ concentration for the impurities were between 98 and 101%. Precision A precision study was also conducted by performing the same procedures at a higher level (150%). The RSD values for the precision study at 100% and 150% concentrations for Impurities 1, 2 and 3 were below 1.0%. The precision study was also conducted at 100% level by performing the same procedures on a different day (inter-day precision). The RSD values of inter-day precision for all impurities were below 2.0%. Linearity and range The calibration equation obtained from regression analysis was used to calculate the corresponding predicted responses. The correlation coefficient for all impurities was more than 0.999; these results are shown in Table III. The coefficient of determination (r 2 ) obtained for Impurity 1, Impurity 2 and Impurity 3 was within the acceptance criteria. Accuracy The percentage recovery values obtained for all impurities were between 98.6 and 102.7%. The percentage recovery values for the impurities were calculated and tabulated in Table IV. Table V Robustness Data: System Suitability Results Sample Parameter 0.5 ml/min 0.9 ml/min 238C 338C ph 5.8 ph Resolution between Impurity 1 and silodosin Tailing factor of the silodosin peak Vali et al.

6 Ruggedness The RSDs were calculated for the areas of each impurity. The RSD for all three impurities was below 1.0%. Table VI Robustness Data: Relative Retention Time of Impurities during Robustness Tests Sample Name 0.5 ml/min 0.9 ml/min 238C 338C ph 5.8 ph Impurity Impurity Impurity Robustness System suitability and 100% spiked sample were injected as per these conditions to determine the resolution of the three impurities. Peak merging was not observed and the resolution between Impurity 1 and silodosin was more than 1.5. The robustness data are shown in Tables V and VI. Solution stability A solution study was performed for sample and system suitability solutions for up to 48 h at room temperature. The solution was found to be highly stable at room temperature during 48 h of Figure 4. Mass spectrum in positive ion ESI mode of (A). Impurity-1, (B). Impurity-2, (C). Impurity-3 and (D). Silodosin. Development of Stability-Indicating UHPLC Method for the Quantitative Determination of Silodosin and Its Related Substances 651

7 Figure 5. Thermo gravimetric thermograms (A) Impurity-1, (B) Impurity-3, (C) Silodosin and (D) Impurity 2. study and no interference/impurities were observed during this study. Discussion Method development and optimization For the determination of silodosin and its impurities, no single analytical stability-indicating UHPLC method is available in the literature. Hence, this study developed a necessary, rapid, accurate and validated method for the simultaneous determination of related substances and degradants. Several trials were conducted by using a PDA detector with different ph values, varied flow rates and different columns and column oven temperatures. Various mobile phase compositions of buffer with organic solvents like acetonitrile and methanol were tried, but the combination of buffer with a small amount of organic phase resulted in a lack of peak symmetry; i.e., tailing more than 2.0 was observed, which is beyond the ICH recommendation of less than 1.5. To minimize this, a few trials were conducted by using triethyl amine as a modifier. The best results were obtained with 100% of 10 mm ammonium acetate buffer mixed with 0.1% triethyl amine, ph 6.0 adjusted with glacial acetic acid. Mobile phase A consisted of 90% buffer and 10% acetonitrile (90:10, v/v) and mobile phase B consisted of 95% acetonitrile with 5% Milli-Q water (95:5, v/v). The Poroshell 120 EC-C18 is an RP packing that can be used for basic, neutral or acidic samples. The exhaustive end capping makes it ideal for use with basic compounds, especially those that produce poor peak shapes on other columns. These columns can be used for a wide range of applications over a ph range of 2 9, accommodating the most popular mobile phases. In the present study, specification of 0.15% was maintained for the three process impurities at 0.5 mg/ml. To confirm the masses of the known process impurities and silodosin, a Thermo Scientific LC MS instrument was used, 0.1% formic acid in 1,000 ml of water used as mobile phase A and 100% methanol was used as mobile phase B. The flow rate was maintained at 0.5 ml/min and 20% of mobile phase A and 80% of mobile phase B were run up to 3 min. The data were collected in positive electrospray ionization (ESI) mode, as shown in Figure 4. Evaluation of potency and relative retention factor Potency evaluation The potency levels of all impurities and API were more than 96.0%. The obtained potency values are shown in Table I and thermograms are shown in Figure 5. The proposed UHPLC method was aimed at developing a chromatographic system that is capable of eluting and resolving silodosin, its three known 652 Vali et al.

8 process impurities and its degradants. The PDA detector is a universal detector that is the most preferred for HPLC and UHPLC analysis in UV-visible region. The potency evaluation for API and impurities is essential, because it provides the exact contents of the compound. Generally, potency evaluation for pharmaceutical impurities is conducted by performing organic tests like HPLC or gas chromatography and inorganic tests like LOD or residue on ignition. Sometimes, because of high costs or a lack of sample quantity in a sufficient amount, these analyses are difficult; for such samples, the TGA technique is the best choice for potency evaluation. This technique is very useful to evaluate potency with 5 to 10 mg of sample in a short run time. This technique involves the loss of weight against temperature. The only limitation is that impurities must be thermally stable. In the present study, silodosin and its impurities were thermally stable. Evaluation of relative retention factor For this evaluation, six solutions containing a mixture of drug substance and impurities were plotted on a graph of concentration versus response for the impurity and standard solutions. The slope of individual linearity plots and the correlation coefficient obtained for impurities and standards was more than The relative response factor (RRF) values are shown in Table I. RRF is an analytical parameter used in chromatographic procedures to control impurities and degradants in drug substances and drug products. RRF is used to correct the differences between the detector responses of impurities and analyte peaks. As per the United States Pharmacopeia (USP), the RRF is the ratio of the responses of equal amounts of the impurities and the drug substance. Conclusion A simple and sensitive RP stability-indicating UHPLC method was developed and validated for the quantitative determination of the process related impurities and degradants of silodosin. Compared with the previously reported methodologies, this was the first specific method for the separation and quantification of related substances of silodosin with UHPLC. The present study will help the manufacturers and suppliers of silodosin to quantify and qualify the purity based on degradation data. Thus, it can be used for routine analysis, quality control and for determining quality during stability studies of pharmaceutical preparations containing related substances. References 1. Yoshida, M., Homma, Y., Kawabe, K.; Silodosin, a novel selective alpha 1A-adrenoceptor selective antagonist for the treatment of benign prostatic hyperplasia; Expert Opinion on Investigational Drugs, (2007); 16: Kazuki, K., Masaki, Y., Yukio, H.; Silodosin, a new a1a-adrenoceptorselective antagonist for treating benign prostatic hyperplasia: results of a phase III randomized, placebo-controlled, double-blind study in Japanese men; British Journal of Urology International, (2006); 98: Masaki, Y., Imao, M., Katsuyoshi, A., Junzo, K.; Silodosin a selective a1a adrenoceptor antagonist for treatment of benign prostatic hyperplasia. In Xianhai, H., Robert, G. (eds). Case studies in modern drug discovery and development. John Wiley & Sons, Somerset, NJ, (2012); Roehrborn, C.G., Male lower urinary tract symptoms (LUTS) and benign prostatic hyperplasia (BPH); Medical Clinics of North America, (2011); 95: Sausville, J., Naslund, M.; Benign prostatic hyperplasia and prostate cancer: an overview for primary care physicians; International Journal of Clinical Practice, (2010); 64: McVary, K.T., Roehrborn, C.G., Avins, A.L.; Update on AUA guideline on the management of benign prostatic hyperplasia; Journal of Urology, (2011); 185: National Institute of Diabetes and Digestive and Kidney Diseases; Prostate enlargement: Benign prostatic hyperplasia, Publication , NIH, Bethesda, (2006). 8. Sharma, C.R., Akhtar, J., Jagani, N.M., Shankharva, Y.R., Shah, J.S.; UV spectrophotometric method for estimation of silodosin from its solid dosage form; Inventi Rapid: Pharm Analysis & Quality Assurance, (accessed December 2, 2012). 9. Chinnalalaiah, R., Ravikumar, P.; Development and validation of RP-HPLC method for estimation of silodosin in bulk and pharmaceutical dosage forms; International Journal of Pharmaceutical Sciences Review and Research, (2012); 16: Matsubara, Y., Kanazawa, T., Kojima, Y., Abe, Y., Kobayashi, K., Kanbe, H., et al.; Pharmacokinetics and disposition of silodosin; Journal of the Pharmaceutical Society of Japan, (2006); 126: Zhao, X., Liu,Y.,Xu, J.,Zhang,D.,Zhou, Y., Gu, J.,et al.; Determination of Silodosin in human plasma by liquid chromatography-tandem mass spectrometry; Journal of Chromatography B, (2009); 877: International Conference on Harmonization (ICH); Q2 (R1), Validation of Analytical Procedures: Text and Methodology, (2005) pdf (accessed December 23, 2010). 13. United States Pharmacopoeia (USP); Validation of Compendial Methods (31st edition). United States Pharmacopeial Convention, Rockville, MD, (2008). 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