HPTLC Method for Determination of Edaravone

HPTLC Method for Deter arcminuteation of EdaravoneDevelopment and Validation of HPTLC Method for Determination of Edaravone in Bulk and in injectable Dosage FormA simple, rapid, reliable and accurate high performance thin layer chromatography method has been developed for the estimation of Edaravone in heap and pharmaceutic dosage form. The chromatographic development was carried out on aluminum plates, pre-coated with silicon oxide gel 60 F254, using a mixture of Toluene Methanol (64v/v) as mobile phase. Detection was carried out densitometrically at 254nm. Thefvalue of analyte was found to be0.660.02. The method was formalize with respect to one-dimensionality, accuracy, precision, limit of detection, limit of quantification and specificity. The linear regression outline data for the calibration plots showed a good linear relationship with 2=0.9995 in the concentration range 200600ng/spot. The % assay (Mean S.D.) was found to be100.270.72. verity of the method was accessed by percentage recovery and found to be99.770.71%. The method is new, simple and economical for routine estimation of edaravone in bulk, pre-formulation studies and pharmaceutical formulation rapidly at low cost in routine analysis.Keywords Edaravone, HPTLC, Pharmaceutical dosage form1. IntroductionEdaravone EDA is a neuroprotective agentused for the purpose of aidingneurologicalrecovery undermentioned acut ebrain ischemia and subsequentcerebral infarction. Chemically, it is 3-Methyl-1-phenyl-2-pyrazolin-5-one. 1. It is a strong novel rationalise radical scavenger, was developed by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). It acts as apotentantioxidant, protect againstoxidative stressandneuronalapoptosis Furthermore, edaravone has anti-apoptotic, anti-necrotic, and anti-inflammatory cytokine effects, as well as scavenging free radicals in cardiovascular diseases and stroke, showing protective effects in the heart, vessel, and brain in experimental studies 2-5 Different me thods have been inform for the determination of EDA in the bulk dose, in the dosage forms and in biological samples. HPLC 6-7 and potentiometric titrations 8 methods are operable for determination of the analyte in bulk medicine and formulation. RP-HPLC 9, RP-HPTLC 10 and LC-MS/MS 11 methods are reported for determination in biological samples.The literature survey revealed that HPTLC method is not reported for determination of EDA in bulk and pharmaceutical dosage forms.The present study describes the development and validation of a simple, specific, sensitive, accurate, precise, and economical HPTLC method for determination of EDA in bulk and injectable dosage form. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines 12,13.Fig 1 Edaravone2. Experimental2.1 Reagents and chemicalsEdaravone was kindly gifted from sun Pharmaceuticals, Vadodara, Gujarat, India. Edaravone injection was obtained from commercial sources at bottom their shelf life period. All the reagents and solvents used were of analytical grade and obtained from Merck Chemicals.2.2. Instrumentation and chromatographic conditionsChromatography was performed on 20cm10cm aluminum foil plates precoated with 0.2mm layers of silica gel 60 F254 (E. Merck, Germany). The plates were prewashed with methanol and water mixture, dried in the current of dry air and activated at 120C for 5min. Samples were applied as bands 6mm wide, by use of a CAMAG (Switzerland) Linomat 5 applicator with a CAMAG microliter syringe. A constant application rate of 150nLs1was employed. Linear ascending development was performed in a twin-trough glass chamberwith mobile phase consisted of toluene methanol (64 v/v), which gave sharp and symmetrical peak withf0.66 + 0.02. The optimized chamber saturation time was 15 min at room temperature (25C2C) and relative humidity60%5%. After development, the plates were dried. Densitometric scanning, at 254 nm, was performe d with a CAMAG TLC scanner 4 in absorbance mode. The source of ray was a deuterium lamp emitting a continuous UV spectrum in the range of 190400 nm.2.3. Preparation of Standard Stock SolutionAn accurately weighed measuring rod of 10mg EDA was transferred to 10mL volumetric flasks, dissolved in methanol, and volume was made up to mark with the same solvent to obtain a working standard having concentration 1000ngL1.2.4. Optimization of mobile phaseInitially, different ratios of methanol and toluene were tried, but tailing of musca volitans was observed. Finally, the mobile phasecomprising of toluenemethanol(64v/v) gives good re issue, sharp and symmetrical peak withvalue of 0.63 at 254nm.Figure 2 Chromatogram of standard Edaravone (Rf = 0.63).3. resolve and discussionValidation of HPTLC methodThe proposed method was validated as per the ICH guidelines in terms of its linearity, accuracy, specificity, intraday and interday precision, limit of detection (LOD), and limit of quantific ation (LOQ).3.1. Linearity (Calibration Curve)The amount of standard solution equivalent to 200-600 ng/spot of EDA was spotted on the prewashed TLC plates. The plates were developed, dried and scanned as described above. The calibration plot was constructed by plotting peak areas against the corresponding concentrations (ng/spot) of EDA. The linearity of response for EDA was assessed in the concentration range 200-600 ng/spot in terms of vend, intercept and correlation coefficient values. The calibration plot showed the correlation coefficient (r2 = 0.999), the intercept (5.838) and the slope (703.3) over the concentration range of 200-600 ng/spot (Fig. 2). The results of regression analysis are shown inTable 1.3.2 Limit of Detection (LOD) and Limit of Quantification (LOQ)The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelinesLOD = 3.3 /SLOQ = 10 /SWhere, = the standard deviation of the response and S = slope of the calibration curve.3.3 RangeSuitable levels of precision and accuracy have been demonstrated between the upper and lour concentration limit of linearity under study.3.4 PrecisionThe intra-day and inter-day variation for the determination of EDA was carried out at three different concentration levels 400, 600, 800 ng/spot. Intra-day variations were assessed by analyzing these concentrations in triplicate within a day and inter-day variation was assessed by using the same concentration of drug and analyzing it different days and time.AccuracyThe accuracy of the method was determined by the use of standard addition at three different levels. The pre analyzed sample solution of 400 ng/spot of EDA was bar with extra amount equivalent to 80 %, 100 % and 120 % of the standard edaravone and the mixtures were analyzed by the proposed me thod. The experiment was conducted in triplicate. When these solutions were analyzed the recoveries were found to be within acceptable limits (Table 1).SpecificityThe mobile phase was optimized and it showed good result. There was no interference of diluents and other constituents in determining peak purity. This method is specific. finishA new HPTLC method has been developed for the identification and quantification of EDA. Low cost, faster speed, and satisfactory precision and accuracy are the main features of this method. The method was successfully validated as per ICH guidelines and statistical analysis proves that the method is sensitive, specific, and repeatable. It can be conveniently employed for routine quality control analysis of EDA as bulk drug and in marketed injectable formulation.AcknowledgmentsThe authors express their gratitude to Sun Pharmaceuticals Vadodara, Gujarat, India for providing a gift sample of Edaravone, the Management of Pioneer Pharmacy Degree College , Vadodara, Gujarat, India, and Anchrom Test lab Pvt. Ltd, Mumbai, Maharastra, India, for providing the necessary facilities.References Nipponese Pharmacopoeial Forum, sixteenth edition, March 2012 Vol.21 (1), pp. 701-702.Doherty, Annette M, Annual Reports in Medicinal Chemistry, Volume 37, Boston Academic Press.Watanabe T, Tanaka M, Watanabe K, Takamatsu Y, Tobe A,Research and development of the free radical scavenger edaravone as a neuroprotectant.Yakugaku Zasshi, March 2004,124(3) 99111.Higashi Y, Jitsuiki D, Chayama K, Yoshizumi M (January 2006). Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a novel free radical scavenger, for treatment of cardiovascular diseases.Recent Patents on Cardiovascular Drug Discovery1(1) 8593.Kikuchi, K. Miura, N. Kawahara, K. Murai, Y. Morioka, M. Lapchak, P. Tanaka, E. Edaravone (radicut), a free radical scavenger, is a potentially useful addition to thrombolytic therapy in patients with acute ischemic stroke (review). Biomed. Rep. 2013, 1, 712.Ge orge Lunn Hplc Methods For Recently Approved Pharmaceuticals A earth-closet Wiley Sons, Inc., Publication P.p 204-206.XIA Ya Jun, ZHANG Xiao Ping Determination of Edaravone Injection by HPLC Chinese Journal of Pharmaceuticals Chinese journal of pharmaceuticals 34 352-353ZHANG Fu-Cheng, TIAN Shu -Xia, JIANG Ye Comparison Of Two Potentiometric Titration Determinations Of edaravone j Chinese Journal Of Pharmaceuticals 2005-09WEI Min, XIAO Yi (Guangxi Liuzhou municipal People s Hospital, Liuzhou 545001, China) Determination of the Concentration of Edaravone in Human Serum by RP-HPLC J China Pharmacy 2007-08M. Gandhimathi, M. Saravana Kumar, R. Baghla and T. K. Ravi RP-HPTLC Method for theIn VitroEstimation of Edaravone in Human PlasmaIndian Pharmaceutical connection Convention Volume 72Issue 2 P.p 276-282GU Li-Qiang XIN Yan-Fei ZHANG Sheng WEN Lei YANG Shi-Bao, HU Xiao-ling, XUAN Yao-Xian Determination of edaravone in plasma of Beagle dog by LC-MS/MS A C 2009ICH-Guidelines Q2A, Valid ation of Analytical Procedures comment and terminology, (CPMP III/5626/94), Geneva, Switzerland, 1995.ICH-Guidelines Q2B, Validation of Analytical Procedures Methodology, (CPMP/ICH/281/95) Geneva, Switzerland 1996.