Stability-indicating HPLC Assay Method and Degradation Profile of Tamsulosin

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1 American-Eurasian Journal of Scientific Research 7 (5): , 2012 ISSN IDOSI Publications, 2012 DOI: /idosi.aejsr Stability-indicating HPLC Assay Method and Degradation Profile of Tamsulosin 1, P.K. Basniwal, S. Panda, S. Jain and Deepti Jain 1 LBS College of Pharmacy, Jaipur , Rajasthan, India 2 Royal College of Pharmacy and Health Sciences, Berhampur, Orissa, India 3 Sagar Institute of Research and Technology, Bhopal, , MP, India 4 School of Pharmaceutical Sciences, Rajiv Gandhi Technological University, Bhopal , MP, India Abstract: A sensitive, stability-indicating liquid-chromatographic method for analysis of tamsulosin in the presence of its degradation products has been developed and validated. Efficient chromatographic separation was achieved on a C18 column with a simple isocratic mobile phase consisting methanol: 50 mm phosphate buffer (ph 7.8, ratio 80:20) at a flow rate of 1 ml/m. The linearity of the method was excellent over the range µg/ml. The method was sensitive, with low limits of detection (10 ng/ml) and quantification (50 ng/ml). The recovery of the method was consistently good ( %) with low (<1%) intra-day and inter-day relative standard deviation. Robustness studies confirmed that peak area was unaffected by small changes in temperature and mobile phase composition. Forced degradation was performed in methanolic solution. In alkaline medium the drug was degraded rapidly as compared to acidic and oxidative condition. The validated, stability-indicating, method was used for analysis of tamsulosin in pharmaceutical dosage form and also to reveal the hydrolytic degradation profile of the tamsulosin. Key words: Tamsulosin Stability-indicating assay HPLC Degradation profile INTRODUCTION and 1D with uroselective 1-blockers like tamsulosin, resulting in the relaxation of the smooth muscles in lower Tamsulosin is an 1A/ 1D subtype selective urinary tract with minimal blood pressure-related adverse 1-adrenoceptor antagonist ( 1-blocker), chemically, effects [3]. (-)(R)-5-(2-(2-(2 ethoxyphenoxy) ethylamino) propyl) -2- Literature survey reveals that determination of methoxybenzene sulfonamide (Fig. 1) and most frequently tamsulosin in various matrixes have been reported as in prescribed medication for the treatment of lower urinary human plasma dialysate, plasma and urine by hightract symptoms suggestive of benign prostatic performance liquid chromatography-electrospray tandem hyperplasia (BPH) [1,2]. The -adrenoceptors mass spectrometry [3], in human plasma using HPLC 1 (,, ) take part in the modulation of blood with fluorescence detection [4], in human plasma by 1A 1B 1D pressure and thus the 1-blockers were originally LC-ESI-MS/MS [5], in dog plasma by LC-MS [6], in developed as antihypertensive agents. Non-selective human plasma by liquid chromatography-electrospray 1-receptor blockage has also additional effects, ionization mass spectrometry [7], in human plasma by including relaxation of the smooth muscles of bladder HPLC using extraction with butyl acetate [8], prostatic neck and prostatic urethra, which are found to relieve and urethral retention of tamsulosin [9], pharmacokinetics the symptoms associated with BPH. Consequently, the and plasma protein binding in rats, dogs and humans first drugs for the treatment of the BPH were non- [10], in human aqueous humor and serum by LC-MS [11], subtype-selective 1-blockers. Unfortunately these by capillary electrophoresis [12], chiral separation by drugs were commonly associated with cardiovascular HPLC [13], related substances of tamsulosin in bulk adverse effects. These side effects were later avoided drugs and formulations [14], spectrophotometrically in by selectively blocking the 1-adrenoceptor subtype s 1A pharmaceutical dosage form [15]. Corresponding Author: Dr. Deepti Jain, School of Pharmaceutical Sciences, Rajiv Gandhi Technological University, Bhopal , MP, India. 193

2 O N H Am-Euras. J. Sci. Res., 7 (5): , 2012 CH 3 OCH 3 SO 2 NH 2 out on a Microbondapak C18 (Lichrospher, Darmstadt, Germany) column of 250 mm 4.6 mm i.d. with particle size of 5µm. Chromatography: Analytes were well eluted in the OC 2 H 5 mobile phase composed of methanol: phosphate buffer Fig. 1: Structure of tamsulosin. (ph 7.8) in the ratio of 80:20 at a flow rate of 1 ml/m. Before use the mobile phase was filtered through a However, in the knowledge authors, no account has Ufipore N 66 Nylon-66, 0.45 µm membrane filter membrane been reported for stability indicating assay method for filter (Pall Life Sciences). All samples were filtered before determination of tamsulosin. The parent drug stability injection (Hamilton 702NR microliter syringe) and test guideline Q1A (R2) issued by International chromatographed at injection volume of 20 µl. The Conference on Harmonization (ICH) [16] suggests that detection wavelength used for tamsulosin and its stress studies should be carried out on a drug to degradation products was 230 nm. establish its inherent stability characteristics, leading to identification of degradation products and hence Method Development: System suitability parameters supporting the suitability of the proposed analytical were optimized after complete saturation of column, six procedures. It also requires that analytical test replicates of HQC (high quality control, 100 µg/ml) of procedures for stability samples should be stability drug were injected separately and column performances indicating and they should be fully validated. like tailing factor, height equivalent to theoretical plates Accordingly, the aims of the present study were to (HETP), retention time (RT) and number of theoretical establish inherent stability of tamsulosin through stress plates were observed. A fresh stock solution (1000 µg/ml) studies under a variety of ICH recommended test of tamsulosin was prepared for calibration curve and six conditions [16-17] and to develop a stability-indicating replicates containing µg/ml were prepared from the assay [18]. stock solution by dilution with the same solvent. Chromatograms were recorded in triplicate for single Experimental dilution and average peak area was plotted against Materials: Tamsulosin was supplied by Dr Reddys concentration. The repeatability of peak area was Laboratory Limited, Hyderabad, India and HPLC grade determined by performing six replicate injections of 10, methanol, water and buffer capsules purchased from 90 and 200 µg/ml solutions to determine intra-day, inter- Merck India Ltd., Mumbai. The other chemicals and day and analyst-to-analyst variation. Apparent recovery solvents used in the studies were of analytical grade, was determined to confirm the suitability and accuracy of from Rankem, Delhi, India. Triple distilled water the method. Six replicates of standard dilutions (50 µg/ml) obtained from Younglin ultra 370 series instrument were analyzed and then spiked with 40, 60 and 80 µg/ml (reverse osmosis of demineralized water) was used of the drug and re-analyzed. To estimate the limits of throughout. detection (LOD) and quantitation (LOQ), diluent was injected six times and the signal-to-noise ratio (S/N) was Instrumentation: Stability studies were carried out in determined. LOD and LOQ were regarded as the amounts humidity chamber (TH 200 S, Thermolab, Germany) and for which S/N was 3:1 and 10:1, respectively. photo stability studies were carried out under the Robustness of the method was studied with sunlight. Sunlight intensity during the studies was variation in mobile phase composition (2%) and column tested using a lux meter (ELM 201, Escon, New Delhi, temperature (5%). The robustness of the method was India). Water baths equipped with temprature controller checked at three different concentrations 40, 60 and 80 (Jindal, S.M. Enterprises, India) were used for hydrolytic µg/ml. The specificity of the method was ascertained by studies. Thermal stability studies were performed in a analyzing drug standard solution and samples of dry air oven (REMI, New Delhi, India). The HPLC system equivalent concentration (50 µg/ml). The identity of the was WATERS 510 model LC system with Turnable 486 peak in the sample was confirmed by comparison of the UV detector. The out put signal was monitored and retention time and UV spectrum of the peak from the 32 processed using millennium software on Pentium sample with those of the peak from the standard. computer. The chromatographic separations were carried Peak purity for the drug was assessed by comparing UV 194

3 spectra acquired at the peak start, peak apex and peak end. It was also ascertained by analyzing standard solution (control solution) and stressed solution of tamsulosin. Analysis of Dosage Form: Powder from tamsulosin tablets/capsules equivalent to 100 mg tamsulosin was sonicated for 30 m with 25 ml methanol to ensure complete extraction of the drug. The volume was then diluted to 50 ml and the solution was filtered through a 0.45 µm membrane filter and diluted to 8 µg/ml for analysis. This experiment was repeated six times. Am-Euras. J. Sci. Res., 7 (5): , 2012 Degradation Studies: Hydrolytic degradation was performed at an initial drug concentration of 1 mg/ml in 0.1 M NaOH/0.1 M Hcl at 60 C for 4 h. Oxidative studies were carried out at room temperature in 3% hydrogen peroxide for 24 h in dark. The pure drug was subjected to photo-degradation by keeping in UV chamber and was kept for 31 h. Additionally, the drug powder was exposed to dry heat at 60ºC for 72 h and to wet heat at 60ºC for 4 h. Samples were withdrawn at Fig. 2: Chromatograms obtained from tamsulosin before appropriate time and subjected to HPLC analysis after forced degradation (A), after treatment with 0.1M suitable dilution. NaOH (B), after treatment with 0.1M HCl (C), after treatment with 3% H2O 2(D). RESULTS AND DISCUSSION Regression equation was found Y = X , The mobile phase was optimized by examining the which was used to calculate the analyte concentration in effect of ph, ionic strength of phosphate buffer and each sample (Table 1). organic modifier (acetonitrile and methanol). The ionic The repeatability of sample injection and strength 50 mm and ph 7.8 for phosphate buffer were measurement of peak area, expressed as RSD (%) are found optimum. At lower ph, capacity factor for the listed in Table 1. Repeatability and intermediate precision degradation product was decreased and some peaks were at three different concentrations (10, 90, 200 µg/ml) for eluted in the void volume. Methanol as organic modifier both within-day and day-to-day analysis were always was found suitable for optimal resolution between the <2%. These low values of the RSD showed the degradation products. A methanol composition of 80 % repeatability and intermediate precision of the method was selected for resolution and shortest chromatographic were good. When the method was used for extraction and run time (8 m) with optimum suitability parameters subsequent analysis of the drug in the dosage form after (Fig 2A). System suitability parameters were analyzed spiking with 50, 100 and 150% of the drug, recovery was to ascertain the system performance consistency for %, standard deviation (Table 1). The LOD number of theoretical plates tailing factor, height for a S/N ratio of 3:1 was 10 ng/ml (RSD 1.56%) and the equivalent to theoretical plates (HETP), retention time LOQ for a signal to noise ratio of 10:1 was 50 ng/ml (RT) and resolution. The % RSD values for these (RSD 1.75%). These results indicated the adequate parameters were found far less than 2%, which indicates sensitivity of the method. Comparison of the retention acceptance of system performance (Table 1). Peak area time and UV spectrum of the peak from the sample with and concentration were subjected to linear least-squares those of the peak from the standard and assessment of regression analysis to calculate the calibration equation peak purity for the drug confirmed the specificity of the and correlation coefficient. The tamsulosin showed good method. Comparison between chromatogram of stressed linearity between µg/ml which was confirmed by the sample and unstressed sample was the clear indication of high value of the correlation coefficient value of the specificity of the method. 195

4 Am-Euras. J. Sci. Res., 7 (5): , 2012 Table 1: Validation parameters for tamsulosin. Parameters a b c Values ± SD, %RSD System Suitability d RT (m) 3.2 ± 0.48, 1.62 Resolution 7.5 ± 1.42, 1.21 No. of Theoretical Plates ± , 1.97 Tailing Factor ± 0.082, 1.37 e HETP (mm) ± 0.83, 1.04 Linearity µg/ml Regression equation Y = X Correlation coefficient 2 r = ± 0.72, 1.74 Precision Repeatability ± 0.319, 0.32 Intermediate precision Inter-day ± 0.47, 0.25 Analyst-to-analyst ± 1.21, 0.12 Accuracy ± 1.17, 0.61 f LOQ 50 ng/ml ± 2.69, 1.75 g LOD 10 ng/ml± 2.27, 1.56 Robustness Mobile phase composition (±2%) ± 2.47, 1.68 Temperature (±5%) ± 1.87, 1.87 Specificity Ascertained by analyzing standard solution (control solution) and stressed solution of tamsulosin a b c d e f mean of six replicates; Standard deviation; % Relative standard deivation; Retention time; Height equivalent to theoretical plates; limit of quantitation; g Limit of detection Table 2: Summary of degradation profile of tamsulosin. Stress conditions Duration of stress condition % remaining of tamsulosin % Mass balance Acidic (0.1 M HCl) 4 h Alkaline (0.1 M NaOH) 4 h Oxidation (3% H2O 2) 24 h Dry heat (40 C) 7 d Dry heat (60 C) 3 d Wet heat (60 C) 24 h UV Light 3 d The drug gradually decreased with time on heating at in light and dark was found to be similar, indicating that 60 C in 0.1 M HCl, forming degradation products at RRT light had no effect on the degradation of the drug in acid 1.6 (relative retention time) (Fig 2B). The rate of and in water. The solid-state studies showed that hydrolysis in acid was slower as compared to that of tamsulosin was stable to the effect of temperature. alkali. About 86 % of drug was found intact after 24 h When the drug powder was exposed to dry heat at 50 C exposure. The drug was found to be more labile to for 7 d and at 60 C for 3 d, no decomposition product of alkaline as compared to acid hydrolysis (Table 2). the drug was observed. Even on exposure to UV light for The reaction in 0.1 M NaOH at 60 C was resulted in 3 d tamsolusin did not show any degradation. about 34% degradation with in 4 h. Drug degradation The drug content in the tablets was quantified using was associated with rise in a major degradation product the proposed analytical method. The absence of at RRT 1.58 m (Fig C). Complete degradation of the drug additional peaks in the chromatogram indicates nonwas observed within 20 h. The drug was moderately interference of the common excipients used in the susceptible to 3% hydrogen peroxide at room temperature tablets. The tablet and capsule were found to be contain (Fig 2D). After 24 h exposure % degradation was not % of the labeled amount of the drug. The low more than 9% (Table 2). No major degradation product coefficient of variation indicates the reproducibility of was observed after exposure of drug solution in water to the assay of tamsulosin in tablet and capsule. It can be sunlight for 2 d only minor degradation products at RRT concluded that the proposed HPLC method is 0.68, 1.6 and 1.98 were formed. The nature of degradation sufficiently sensitive and reproducible for the analysis 196

5 % of Tamsulosin % of Tamsulosin Hydorlytic degradation Am-Euras. J. Sci. Res., 7 (5): , 2012 [A] as the bulk drug and in the pharmaceutical dosage form and other quality control samples obtained during product development. REFERENCES 0 1. Dunn, C.J., A. Matheson and D.M. Faulds, Tamsulosin: A Review of its Pharmacology and Time in minutes Therapeutic Efficacy in the Management of Lower Alkaline degradation Acidic degradation Urinary Tract Symptoms. Drugs Aging, 19: Oxidative degradation [B] 2. Chapple, C. and K.E. Andersson, Tamsulosin: An Overview. World J. Urol., 19: Matsushima, H., K.I. Takanuki, H. Kamimura, 98 T. Watanabe and S. Higuchi, Highly Sensitive 96 Method for the Determination of Tamsulosin Hydrochloride in Human Plasma Dialysate, Plasma 90 and Urine by High-Performance Liquid 88 Chromatography-Electrospray Tandem Mass Time in hours spectrometry.j. Chromatogr B., 695: Soeishi, Y., M. Kobori, S. Kobayashi and S. Higuchi, Fig. 3: Hydrolytic profile (A) and oxidative profile (B) of Sensitive Method for the Determination of tamsulosin. Tamsulosin in Human Plasma Using Highof Performance Liquid Chromatography with tamsulosin in pharmaceutical dosage forms within a Fluorescence Detection. J. Chromatogr., 533: 291. short analysis time. No degradation was observed on 5. Ramakrishna, N.V., K.N. Vishwottam, S. Manoj, HPLC analysis of marketed tablets subjected to thermal M. Koteshwara, S. Wishu and D.P. Varma, stress at 50ºC and separately to an accelerated condition Rapid, Simple and Highly Sensitive LC-ESI-MS/MS of 40ºC/75% RH (relative humidity) for 3 months. It was Method for the Quantification of Tamsulosin in shown that a single degradation product (RT = 3.2 m) Human Plasma. Biomed. Chromatogr., 19: 709. was formed on exposure of the formulation to light. 6. Qi, M., P. Wang and L. Liu, Determination of The study indicated that the method could be applied Tamsulosin in Dog Plasma by Liquid Chromatography successfully even to stress marketed formulation. with Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry.J. Chromatogr. B., CONCLUSION 805(1): Ding, L., L. Li, P. Tao, J. Yang and Z. Zhang, A new, sensitive and stability-indicating LC method Quantitation of Tamsulosin in Human Plasma by for analysis of tamsulosin has been successfully Liquid Chromatography-Electrospray Ionization Mass developed. Simple isocratic elution and an easy extraction Spectrometry. J. Chromatogr. B., 767(1): procedure enabled rapid and cost-effective analysis of 8. Macek, J., J. Klima and P. Ptacek, the drug. The method was accurate and precise with Rapid Determination of Tamsulosin in Human Plasma consistently good recovery. It also enabled good by High-Performance Liquid Chromatography Using resolution of the drug from its degradation products, Extraction with Butyl acetate. J. Chromatogr. B., impurities and formulation excipients. The drug was 809: 307. rapidly degraded under alkaline conditions as compared 9. Sato, S., A. Ohtake, H. Matsushima, C. Saitoh, to acidic medium, oxidative condition while it was found S. Usuda and K. Miyata, Pharmacological Effect to be stable in dry heat, wet heat and UV light. of Tamsulosin in Relation to Dog Plasma and Tissue Thus, tamsulosin was susceptible to hydrolytic and Concentrations: Prostatic and Urethral Retention oxidative degradation. The validated stability-indicating Possibly Contributes to Uroselectivity of Tamsulosin. method can be used for routine analysis of tamsulosin J. Pharmacol. Exp. Ther., 296:

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