HPTLC Method for Determination of Edaravone

HPTLC method for Determination of EdaravoneDevelopment and validation of HPTLC Method for Determination of Edaravone in Bulk and in Injectable social disease FormA simple, rapid, reliable and accurate high performance svelte layer chromatography system has been genuine for the estimation of Edaravone in bulk and pharmaceutic dosage form. The chromatographic development was carried out on aluminum plates, pre-coated with silicon dioxide gel 60 F254, using a mixture of Toluene wood alcohol (64v/v) as mobile phase. Detection was carried out densitometrically at 254nm. Thefvalue of analyte was rear to be0.660.02. The order was authorise with respect to analogity, verity, precision, leap of detection, demarcation of quantification and specificity. The analogue regression epitome data for the calib symmetryn plots showed a good linear relationship with 2=0.9995 in the parsimony range 200600ng/ turn. The % assay (Mean S.D.) was found to be100.270.72. Accuracy of the rule was accessed by percentage recuperation 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 instrumentused for the purpose of aidingneurologicalrecoveryfollowing acut ebrain ischemia and ulteriorcerebral infarction. Chemically, it is 3-Methyl-1-phenyl-2-pyrazolin-5-one. 1. It is a strong novel free antecedent scavenger, was demonstrable by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). It acts as apotentantioxidant, protecting against aerophilic 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 st udies 2-5 Different methods have been reported for the aim of EDA in the bulk drug, in the dosage forms and in biological samples. HPLC 6-7 and potentiometric titrations 8 methods be available for determination of the analyte in bulk drug and formulation. RP-HPLC 9, RP-HPTLC 10 and LC-MS/MS 11 methods ar 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 indoors their shelf life period. All the reagents and solvents used were of analytical grade and obtained from Merck Chemicals.2.2. orchestration 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, dry out in the current of dry air out and activated at cxxC for 5min. Samples were applied as bands 6mm wide, by use of a CAMAG (Switzerland) Linomat 5 applicator with a CAMAG microliter syringe. A incessant application rate of 150nLs1was employed. Linear ascending development was performed in a twin-trough glass chamberwith mobile phase consisted of methylbenzene methanol (64 v/v), which gave sharp and symmetrical stage 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 performed with a CAMAG TLC scanner 4 in absorbance mode. The source of radiation was a deuterium lamp emitting a continuous UV spectrum in the range of 190400 nm.2.3. Preparation of Standard commonplace SolutionAn accurately weighed quantity of 10mg EDA was transferred to 10mL volumetric flasks, dissolved in methanol, and volume was made up to cross off with the said(prenominal) solvent to obtain a working standard having concentration 1000ngL1.2.4. Optimization of mobile phaseInitially, dissimilar ratios of methanol and toluene were tried, but tailing of blots was observed. Finally, the mobile phasecomprising of toluenemethanol(64v/v) gives good re closure, sharp and symmetrical peak withvalue of 0.63 at 254nm.Figure 2 Chromatogram of standard Edaravone (Rf = 0.63).3. Result and sermon validation of HPTLC methodThe proposed method was validated as per the ICH guidelines in foothold of its linearity, accuracy, specificity, intraday and interday precision, limit of detection (LOD), and limit of quantification (LOQ).3.1. Linearity (Calibration Curve)The amount of standard solution equivalent to 200-600 ng/ be intimate 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 beas against the agree concentrations (ng/spot) of EDA. The linearity of retort for EDA was assessed in the concentration range 200-600 ng/spot in terms of slope, intercept and coefficient of 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 LO D 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 lower concentration limit of linearity under study.3.4 PrecisionThe intra-day and inter-day variation for the determination of EDA was carried out at threesome different concentration levels 400, 600, 800 ng/spot. Intra-day variations were assessed by analyzing these concentrations in reproduce 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 spiked with extra amount equivalent to 80 %, 100 % and 120 % of the sta ndard edaravone and the mixtures were analyzed by the proposed method. 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. on that point was no interference of diluents and other constituents in find peak purity. This method is specific.ConclusionA 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 caliber 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 gif t sample of Edaravone, the Management of initiate Pharmacy Degree College, Vadodara, Gujarat, India, and Anchrom Test lab Pvt. Ltd, Mumbai, Maharastra, India, for providing the necessary facilities.ReferencesJapanese Pharmacopoeial Forum, ordinal edition, March 2012 Vol.21 (1), pp. 701-702.Doherty, Annette M, Annual Reports in Medicinal Chemistry, Volume 37, Boston faculty member 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 clot buster therapy in patients with acute ischemic stroke (review). Biomed. 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