WORLD JOURNAL OF PHARMACEUTICAL AND MEDICAL RESEARCH

wjpmr, 2016,2(5), 122-127. SJIF Impact Factor: 3.535 WORLD JOURNAL OF PHARMACEUTICAL AND MEDICAL RESEARCH www.wjpmr.com Research Article ISSN 2455-3301 WJPMR DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING HPTLC METHOD FOR DETERMINATION OF ACEBROPHYLLINE Ajinkya A. Deosthali and Mrinalini C. Damle * AISSMS College of Pharmacy, Kennedy Road, Near Rto, Pune-411001. *Corresponding Author: Dr. Mrinalini C. Damle AISSMS College of Pharmacy, Kennedy Road, Near Rto, Pune-411001. Article Received on 17/06/2016 Article Revised on 05/07/2016 Article Accepted on 26/07/2016 ABSTRACT A simple, rapid validated stability indicating HPTLC method of Acebrophylline was successfully developed. This method is based on HPTLC separation afollowed by UV detection at 248 nm and 273 nm. The separation was carried out on merck TLC aluminium sheets precoated with silica gel 60F254 using toluene: methanol (5:5 v/v) as a mobile phase. Acebrophylline gave well defined and sharp peak at Rf 0.48 ± 0.02 at 273 nm and 0.85±0.02 at 248 nm. Calibration curve was linear in range 500-2500 ng/band. Stress degradation study shows that sample degraded with acid and base hydrolysis, under oxidation, thermal and photolytic stress conditions. The peak purity parameter ensured non-interference by product of degradation. This method can be applied to determination of stability of Acebrophylline. The suitability of this HPTLC method for quantitative determination of Acebrophylline was proved by validation in accordance with requirements of ICH guidelines. KEYWORDS: Acebrophylline, HPTLC, Stability, Validation. INTRODUCTION Acebrophylline is chemically 4-[(2-amino-3,5 dibromophenyl)methylamino]cyclohexan-1-ol;2-(1,3- dimethyl-2,6-dioxopurin-7-yl)acetic acid. It is used as Bronchodilator in treatment of asthma. [1] Literature survey revealed that various analytical methods like spectrophotometric [2-3], RP-HPLC [4-7], LC-MS [8], simple TLC [9], SIM- HPLC [10-13] have been reported for the determination of Acebrophylline, but no stability indicating HPTLC method has yet been reported for estimation of Acebrophylline. Development of SIM is based on systematic exposure of API to various stress conditions. Systematic optimization trials are required to arrive at combination of concentration of stress reagent and duration of exposure, to obtain degradation preferably in the 10-30% range. Typical degradative conditions involve hydrolysis under different ph conditions, photolysis, oxidation and thermal. MATERIALS AND METHODS Reagents and Chemicals Working standard of Acebrophylline were obtained from SAVI PHARMA, Majhitar, Sikkim(India). Methanol (AR grade), Toluene (AR grade), Hydrochloric acid (HCl), hydrogen peroxide (H 2 O 2, 30% v/v), sodium hydroxide (NaOH) were purchased from LOBA CHEMIE PVT. LTD. Mumbai. Preparation of Standard Stock Solution Standard stock solution of Acebrophylline were prepared by dissolving 10 mg of drug in 10 ml of methanol get concentration of 1000 µg/ml. From the standard stock solution, working standard solution was prepared containing 100µg/ml of Acebrophylline using Methanol. Selection of Detection Wavelength From the standard stock solution further dilutions were done using methanol and scanned over the range of 200-400 nm and the spectra was obtained. It was observed that Acebrophylline showed considerable absorbance at 248 nm and 273 nm. Fig 1: Structure of Acebrophylline. www.wjpmr.com 122

Oxidation Degradation µg/ml) was mixed with 1ml 30% v/v H 2 O 2, volume was made up to 10ml with Methanol. Solution was kept for 1/2hr and applied on TLC plate. Degradation Under Dry Heat Dry heat study was performed by keeping both drugs in oven at 60 0 C. A sample was withdrawn after 4hrs, weighed and dissolved in methanol to get solution of 100µg/ml of Acebrophylline and then applied on TLC plate. Fig 2: Absorbance Spectrum of Acebrophylline (10 µg/ml). Chromatographic state and Instrumentation Chromatographic separation of drug was performed on Aluminum plates precoated with silica gel 60 F 254, (10 cm 10 cm with 250 µm layer thickness).samples were applied on the plate as a band with 5 mm width using Camag 100 μl sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland). The mobile phase was composed of Toluene: Methanol (5:5 v/v).10 cm 10 cm CAMAG twin trough glass chamber was used for linear ascending development of TLC plate under 15 min saturation conditions and 10 ml of organic solvent was used per run, migration distance was 90 mm. Densitometric scanning was performed using Camag TLC scanner 3 in the range of 400-200 nm, operated by wincats software (Version 1.4.3, Camag), slit dimensions were 3.00 x 0.45 mm and Deuterium lamp was used as a radiation source. Photo-Degradation Photolytic studies were carried out by exposure of drug to UV light up to 200 watt hrs/square meter and subsequently to cool fluorescent light to achieve an illumination of 1.2 million Lux hrs. Sample was weighed, dissolved and diluted get 100 µg/ml as final concentration and was applied on TLC plate. RESULT AND DISCUSSION Optimization of Chromatographic Conditions The primary objective in developing this stability indicating HPTLC method is to achieve the resolution of Acebrophylline. The chromatographic separation was achieved by linear ascending development in 10 cm 10 cm twin trough glass chamber using. Toluene: Methanol (5:5 v/v) as mobile phase and detection was carried out at 248 nm and 273 nm. The retention factor for Acebrophylline was found to be 0.85 ± 0.02 at 248 nm and 0.48 at 273 nm. Representative Densitogram of standard solution of Acebrophylline is shown in figure. Stress Degradation Studies of Bulk Drug Stress testing studies were carried out to provide evidence on how the quality of drug varies under the influence of variety of environmental conditions like hydrolysis under different ph conditions, oxidation, heat etc. Alkali Catalyzed Hydrolysis µg/ml) was mixed with 1ml of 0.1 N NaOH (methanolic) and volume was made up to 10ml with methanol. Solution was kept for 1/2 hr and applied on TLC plate. Acid Catalyzed Hydrolysis µg/ml) was mixed with 1 ml of 0.1 N HCl (methanolic) and volume was made up to 10ml with methanol. Solution was kept for 1 hr and applied on TLC plate. Neutral Hydrolysis µg/ml) was mixed with 1 ml water, volume was made up to 10 ml with methanol. Solution was kept for 1 hr. and applied on TLC plate. www.wjpmr.com 123

Fig. 3: Densitogram of standard solution of Acebrophylline at 248 nm at 273. Result of Forced Degradation Studies Degradation was observed for Acebrophylline during stress conditions like hydrolysis, oxidation, dry heat and light. Results of the stress degradation studies are presented in Table 1. No peak was observed for product of any reagent induced degradation. Table 1: Summary of stress degradation of Acebrophylline. At 248 nm At 273 nm Peak purity Percent Percent Percent Percent at 248 nm at 273 Stress degradation conditions recovery degraded recovery degraded r(s,m) r(m,e) r(s,m) r(m,e) (%) (%) (%) (%) Initial 100-100 - 0.9995 0.9993 0.9992 0.999 Base (0.1 N NaOH, kept for 1/2 hr) 84.65 15.35 87.41 12.59 0.9994 0.9992 0.9993 0.9998 Acid (0.1 N HCl, Kept for 1 hr) 80.43 19.57 84.10 15.9 0.9996 0.9993 0.9992 0.9992 H 2 O 2 30% v/v 80.29 19.71 80.52 19.48 0.9997 0.9994 0.9998 0.9997 (kept for 1/2 hr) Neutral (kept for 1 hr) 77.46 22.54 77.23 22.77 0.9993 0.9996 0.9997 0.9994 Heat dry (60 0 C, 4 hrs) 86.05 13.95 87.03 12.97 0.9992 0.9995 0.9998 0.9997 Photo stability UV, 200 watt hrs/square meter Florescence, 1.2 million Lux. Hrs 68.85 58.54 31.15 41.46 68.42 51.29 31.58 48.71 0.9995 0.9994 0.9993 0.9992 0.9992 0.9998 0.9990 0.9994 www.wjpmr.com 124

Validation of The Method The method was validated for various parameters in accordance with ICH guidelines. Linearity The calibration curve was obtained in the range of 500-2500 ng/band for Acebrophylline by applying different volumes on TLC of stock solution 100 μg/ml resp. Each standard in six replicates was analyzed and peak areas were recorded. Standard calibration graph was plotted of peak area Vs concentration applied. The equation of the calibration curve found for Acebrophylline was y= 2.4612x +787.4 at 248 nm and y= 4.145x+ 1521.6 at 273 nm.the coefficient of correlation (r 2 ) was found to be 0.9905 and 0.9939 for Acebrophylline at 248 nm and 273 respectively shown in Figure. Fig. 4: Densitogram of linearity of Acebrophylline at 248 nm and at 273nm. Fig 5: Calibration curve for Acebrophylline at 248 nm and 273 nm Precision The precision of the system was demonstrated by intraday and inter-day studies. In the intraday study 6 replicates of 1 standard concentrations (1500ng/band for Acebrophylline) were analyzed in a day and percentage RSD was calculated. For the inter day study, 3 replicates of 3 consecutive concentrations were analyzed and percentage RSD was calculated. For interday system precision 0.42 % and 0.56 % at 248 nm and 273 nm respectively. For intraday RSD was found to be 0.62 and 1.78% for Acebrophylline at 248 nm and 273 nm respectively. Assay Five tablets (Marketed product) each containing 100 mg of Acebrophylline were weighed and powdered. Powder equivalent to 10 mg of drug was transferred to 10 ml volumetric flask and was diluted with methanol, sonicated for 10 min and volume made to 10 ml (1000 µg/ml). Solution was filtered through Whatman filter paper and further dilutions were made to get the final concentration of 100 µg/ml of which 10 µl volumes was applied on plate. Analysis was repeated for two times. % assay was determined from linearity equation. Table 2: Results of Assay of Acebrophylline. % Recovery Lable at 248 at 273 claim nm nm 100 mg 1000 98.94 101.66 Accuracy To check accuracy of the method, recovery studies were carried out by adding standard drug to sample at three different levels 80, 100 and 120 %. Basic concentration of sample was 1000 ng/band from Marketed tablet of Acebrophylline. The drug concentrations were calculated from respective linearity equation. The results of the recovery studies indicated that the method is accurate for estimation of drug in tablet. The results obtained are shown in. www.wjpmr.com 125

Table 3: Recovery studies of Acebrophylline at 248 nm and 273 nm. Level (%) Std. Sample Area Recovered % Recovery 80 800 1000 5313.3 1839.83 102.21 100 1000 1000 5742.8 2014.22 100.71 120 1200 1000 6213.7 2205.69 100.25 Level (%) Std. Sample Area Recovered % Recovery 80 800 1000 9394.4 1837.2 102.06 100 1000 1000 9838 2008.93 100.44 120 1200 1000 10291.7 2174.3 98.81 Specificity The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be more than 0.9992, indicating the noninterference of any other peak of degradation product or impurity. Limit of Detection (LOD) AND Limit of Quantitation (LOQ) LOD and LOQ were calculated as 3.3 σ/s and 10 σ/s, respectively; where σ is the standard deviation of the concentration response (y-intercept) and S is the slope of the calibration plot. The LOD and LOQ were found to be. At 248 nm LOD of Acebrophylline=44.16 ng/band LOQ of Acebrophylline= 133.82ng/band At 273 nm LOD of Acebrophylline= 36.67ng/band LOQ of Acebrophylline= 111.12 ng/band Robustness Robustness of the method was determined by carrying out the analysis under conditions during which mobile phase ratio, chamber saturation time were altered, Time was also changed from spotting to development and development to scanning and the effects on the peak area was noted. Table 4: Robustness Study. Sr. No. 1. 2. 3 4 Parameters Saturation time (15min) ± 2 min. Mobile phase composition Toluene: Methanol (5:5 v/v) ±0.2 Time from spotting to development (immediate) Time from development to scanning (immediate) % RSD Robust condition At 248nm At 273nm 13min 1.50 0.75 17min 1.41 0.78 Toluene: Methanol(5.2: 4.8 v/v) 0.89 0.29 Toluene: Methanol (5.8: 5.2 v/v) 0.65 1.28 After 30min. 1.21 1.77 After 1hr 1.54 0.98 After 30min. 1.30 1.9 After 1hr 174 1.88 Table 5: Summary of validation study. Sr. No. Validation parameters Linearity Equation 1. (r2) Range 2. Precision (% RSD) Acebrophylline at 248 nm Rf=0.85 at 273 nm Rf= 0.48 y= 2.4612x + 787.4 y = 4.145x + 1521.6 R² = 0.9905 R² = 0.9939 500-2500 ng/band 500-2500 ng/band www.wjpmr.com 126

Interday Intraday 0.42 0.62 0.56 1.78 Accuracy 3. 80% 102.21 102.06 100% 100.71 100.44 120% 100.25 98.81 4. Limit of Detection 44.16 36.37 5. Limit of Quantitation 133.82 111.12 6. Specificity Specific 7. Robustness Robust CONCLUSION The method developed is simple, rapid and stability indicating. It may be used to monitor stability of Acebrophylline. REFERENCES 1. https://www.google.co.in/acebrophylline (accessed on 18/05/2016). 2. Tvinkal P. Patel et al. Q-absorbance ratio method for simultaneous estimation of Acetylcysteine and Acebrophylline. World Journal of Pharmaceutical Research., 2015; 4(5): 1808-1816. 3. Jignesh Maniya et al. Development and Validation of Spectroscopic Method for Simultaneous Estimation of Acebrophylline and Montelukast Sodium in Combined Dosage Form. Indo American Journal of Pharmaceutical Research., 2012; 2(10): 1027-1036. 4. A. Geetha susmita et al. Simultaneous estimation of Acebrophylline and Acetylcysteine in tablet dosage form by RP HPLC method. Asian journal of pharmaceutical research., 2015; 5(3): 143-150. 5. Sridhar Thota et al. RP-HPLC Analysis of Acebrophylline in API and Capsule Dosage Form. Research Journal of Pharmaceutical, Biological and Chemical Sciences., 2014; 5(1): 480-486. 6. S. Ramanjaneyulu et al. Development and validation of RP- HPLC method for the estimation of Acebrophylline in capsules. International Journal of Inventions In Pharmaceutical Sciences., 2013; 1(5): 404-408. 7. Mohit R. Bauskar, Development and Validation of Reverse Phase Liquid Chromatographic Methods for the Determination of Acebrophylline in Capsule Form, Research Journal of Pharmacy and Technology, 2011; 4(10): 1542-1546. 8. Kyung-Don Nam et al. Bioequivalence Assessment of Acephyll Capsule to Surfolase Capsule (Acebrophylline HCl 100 mg) by Liquid Chromatography Tandem Mass Spectrometry. Journal of Pharmaceutical Investigation., 2011; 41(5): 309-315. 9. W.D. Sam Solomon et al. Application of TLC - Densitometry method for estimation of Acebrophylline in pharmaceutical dosage forms. Journal of Pharmacy Research., 2010; 3(11): 2561-2563. 10. Hitesh. J. Vekaria et al. Analytical Method Development and Validation for Simultaneous Estimation of Acebrophylline and Montelukast Sodium in their Pharmaceutical Dosage Form. Journal of Pharmaceutical science and bioscientific research., 2015; 5(5): 475-480. 11. Sunil R Dhaneshwar et al, Development and Validation of Stability Indicating RP-HPLC-PDA Method for Determination of Acebrophylline and It s Application for Formulation Analysis and Dissolution Study, Journal of Basic and Applied Scientific Research, 2011; 1(11): 1884-1890. 12. Thesia DU et al, Stability Indicating HPLC Method Development for Estimation of Montelukast Sodium and Acebrophylline in Combined Dosage Form, Journal club for Pharmaceutical Sciences, 2014; 1(1): 99-114. 13. Krishna R Gupta et al, Stability Indicating HPLC Assay Method Development for determination of Acebrophylline, Inventi Rapid - Pharm Analysis & Quality Assurance, 2013; 2013(1): 790-798. www.wjpmr.com 127