HPTLC Method for Determination of Edaravone

HPTLC Method for Determination of Edaravone Development and Validation of HPTLC Method for Determination of Edaravone in Bulk and in Injectable Dosage Form A simple, rapid, reliable and accurate high performance thin layer chromatography method has been developed for the estimation of Edaravone in bulk and pharmaceutical dosage form. The chromatographic development was carried out on aluminum plates, pre-coated with silica gel 60 F254, using a mixture of Toluene: Methanol (6:à ¢Ã¢â€š ¬Ã¢â‚¬ °4à ¢Ã¢â€š ¬Ã¢â‚¬ °v/v) as mobile phase. Detection was carried out densitometrically at 254à ¢Ã¢â€š ¬Ã¢â‚¬ °nm. Theà °Ã‚ Ã¢â‚¬ËœÃ¢â‚¬ ¦fvalue of analyte was found to be0.66 ±0.02. The method was validated with respect to linearity, accuracy, precision, limit of detection, limit of quantification and specificity. The linear regression analysis data for the calibration plots showed a good linear relationship with Ã… ¸2=0.9995 in the concentration range 200–600à ¢Ã¢â€š ¬Ã¢â‚¬ °ng/spot. The % assay (Mean  ± S.D.) was found to be100.27 ±0.72. Accuracy of the method was accessed by percentage recovery and found to be99.77 ±0.71%. The m ethod 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 form 1. Introduction Edaravone [EDA] is a neuroprotective agent  used for the purpose of aiding  neurological  recovery  following acut ebrain ischemia and subsequent  cerebral infarction. Chemically, it is 3-Methyl-1-phenyl-2-pyrazolin-5-one. [1]. It is a strong novel free radical scavenger, was developed by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). It acts as a  potentantioxidant, protecting against  oxidative stress  and  neuronal  apoptosis 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 methods have been reported for the determination of EDA in the bulk drug, in the dosage forms and in biological samples. HPLC [6-7] and potentiometric titrations [8] methods are available for determination of the analyte in bulk drug 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: Edaravone 2. Experimental 2.1 Reagents and chemicals Edaravone was kindly gifted from Sun Pharmaceuticals, Vadodara, Gujarat, India. Edaravone injection was obtained from commercial sources within their shelf life period. All the reagents and solvents used were of analytical grade and obtained from Merck Chemicals. 2.2. Instrumentation and chromatographic conditions Chromatography was performed on 20cmÃâ€"10cm 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 120 °C 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 150nLs−1was employed. Linear ascending development was performed in a twin-trough glass chamberwith mobile phase consisted of toluene: methanol (6:4 v/v), which gave sharp and symmetrical peak withà °Ã‚ Ã¢â‚¬ËœÃ¢â‚¬ ¦f0.66 + 0.02. The optimized chamber saturation time was 15 min at room temperature (25à ¢Ã‹â€ Ã‹Å"C ±2à ¢Ã‹â€ Ã‹Å"C) 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 cont inuous UV spectrum in the range of 190–400 nm. 2.3. Preparation of Standard Stock Solution An accurately weighed quantity of 10à ¢Ã¢â€š ¬Ã¢â‚¬ °mg EDA was transferred to 10à ¢Ã¢â€š ¬Ã¢â‚¬ °mL volumetric flasks, dissolved in methanol, and volume was made up to mark with the same solvent to obtain a working standard having concentration 1000à ¢Ã¢â€š ¬Ã¢â‚¬ °ngà ¢Ã¢â€š ¬Ã¢â‚¬ °ÃŽ ¼L−1. 2.4. Optimization of mobile phase Initially, different ratios of methanol and toluene were tried, but tailing of spots was observed. Finally, the mobile phasecomprising of toluene:à ¢Ã¢â€š ¬Ã¢â‚¬ °methanolà ¢Ã¢â€š ¬Ã¢â‚¬ °(6:à ¢Ã¢â€š ¬Ã¢â‚¬ °4à ¢Ã¢â€š ¬Ã¢â‚¬ °v/v) gives good resolution, sharp and symmetrical peak withà °Ã‚ Ã¢â‚¬ËœÃ¢â‚¬ ¦Ãƒ °Ã‚ Ã‚ Ã‚ ¹value of 0.63 at 254à ¢Ã¢â€š ¬Ã¢â‚¬ °nm. Figure 2: Chromatogram of standard Edaravone: (Rf = 0.63). 3. Result and discussion Validation of HPTLC method: The 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 quantification (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 slope, 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) guidelines LOD = 3.3 Ãâ€" ÏÆ'/S LOQ = 10 Ãâ€" ÏÆ'/S Where, ÏÆ' = the standard deviation of the response and S = slope of the calibration curve. 3.3 Range Suitable levels of precision and accuracy have been demonstrated between the upper and lower concentration limit of linearity under study. 3.4 Precision: The 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. Accuracy: The 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 standard 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). Specificity The mobile phase was optimized and it showed good result. There was no interference of diluents and other constituent’s in determining peak purity. This method is specific. Conclusion A 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. Acknowledgments The 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 Japanese 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,â€Å"Research and development of the free radical scavenger edaravone as a neuroprotectant.Yakugaku Zasshi, March 2004,124(3): 99–111. 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): 85–93. 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, 7–12. George Lunn Hplc Methods For Recently Approved Pharmaceuticals A John 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; 352-353 ZHANG Fu-Cheng, TIAN Shu -Xia, JIANG Ye Comparison Of Two Potentiometric Titration Determinations Of edaravone [j]; Chinese Journal Of Pharmaceuticals; 2005-09 WEI 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; 2007-08 M. Gandhimathi, M. Saravana Kumar, R. Baghla and T. K. Ravi RP-HPTLC Method for theIn VitroEstimation of Edaravone in Human PlasmaIndian Pharmaceutical Association Convention Volume: 72Issue: 2 P.p: 276-282 GU 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]; 2009 ICH-Guidelines Q2A, â€Å"Validation of Analytical Procedures: Definition and terminology,† (CPMP III/5626/94), Geneva, Switzerland, 1995. ICH-Guidelines Q2B, â€Å"Validation of Analytical Procedures: Methodology,† (CPMP/ICH/281/95) Geneva, Switzerland 1996.

Porter’s 5 Forces Analysis Essay

Threat of New Entrants. The average person can’t come along and start up a bank, but there are services, such as internet bill payment, on which entrepreneurs can capitalize. Banks are fearful of being squeezed out of the payments business, because it is a good source of fee-based revenue. Another trend that poses a threat is companies offering other financial services. What would it take for an insurance company to start offering mortgage and loan services? Not much. Also, when analyzing a regional bank, remember that the possibility of a mega bank entering into the market poses a real threat. Power of Suppliers. The suppliers of capital might not pose a big threat, but the threat of suppliers luring away human capital does. If a talented individual is working in a smaller regional bank, there is the chance that person will be enticed away by bigger banks, investment firms, etc. Power of Buyers. The individual doesn’t pose much of a threat to the banking industry, but one major factor affecting the power of buyers is relatively high switching costs. If a person has a mortgage, car loan, credit card, checking account and mutual funds with one particular bank, it can be extremely tough for that person to switch to another bank. In an attempt to lure in customers, banks try to lower the price of switching, but many people would still rather stick with their current bank. On the other hand, large corporate clients have banks wrapped around their little fingers. Financial institutions – by offering better exchange rates, more services, and exposure to foreign capital markets – work extremely hard to get high-margin corporate clients. Availability of Substitutes. As you can probably imagine, there are plenty of substitutes in the banking industry. Banks offer a suite of services over and above taking deposits and lending money, but whether it is insurance, mutual funds or fixed income securities, chances are there is a non-banking financial services company that can offer similar services. On the lending side of the business, banks are seeing competition rise from unconventional companies. Sony (NYSE: SNE), General Motors (NYSE:GM) and Microsoft (Nasdaq:MSFT) all offer preferred financing to customers who buy big ticket items. If car companies are offering 0% financing, why would anyone want to get a car loan from the bank and pay 5-10% interest? Competitive Rivalry. The banking industry is highly competitive. The financial services industry has been around for hundreds of years, and just about everyone who needs banking services already has them. Because of this, banks must attempt to lure clients away from competitor banks. They do this by offering lower financing, preferred rates and investment services. The banking sector is in a race to see who can offer both the best and fastest services, but this also causes banks to experience a lower ROA. They then have an incentive to take on high-risk projects. In the long run, we’re likely to see more consolidation in the banking industry. Larger banks would prefer to take over or merge with another bank rather than spend the money to market and advertise to people.

The Purpose of the Sub-plot in Shakespeare’s Twelfth Night

Twelfth Night or What You Will is one of Shakespeare’s most famous comedies. It has been performed hundreds of times and adapted into a number of modern films. The main plot of the play follows Viola, a girl who is rescued from a shipwreck and enters into the service of the Duke Orsino disguised as a man. Rising quickly in his estimation, Viola begins delivering messages of love on his behalf to Olivia, a noble woman who has no interest in Orsino’s advances. Over the course of the play Olivia falls in love with the disguised Viola, Viola falls in love with Orsino, and Viola’s twin brother Sebastian, who supposedly died in the shipwreck, returns. Following Sebastian’s return the twins are mistaken for each other, leading to both†¦show more content†¦Therefore, both plots have symmetrical patterns with slight differences. In each plot, Olivia is the object of men’s affections and in both plots the men trying to woo her are thwarted. Salingar also argues that the sub-plot serves to amplify the main themes of the play. He asserts that the theme of misrule in the comic subplot â€Å"gives the underlying constructive principle of the whole play,† (Salingar 118) and the elements of unconscious parody in the sub-plot help to reinforce the theme of delusion and error. Misrule is common to both plots, perhaps more directly in the sub-plot and indirectly in the main plot. In the main plot the setting is fantasy-like, every day is a feast and the aristocracy enjoys freedom from the normal social constraints that characterized the period in which Shakespeare was writing. Misrule is necessary to explain a number of points in the main plot such as the sudden switch of romantic partners at the end of the play and Viola’s ability to remain undetected as a woman despite her feeble attempts at concealment. She nearly admits it to Olivia in the scene where she says, â€Å"What I am, and what I would, are as secret as maidenhead (1.5.). According to Cahill the subplot is, â€Å"historically-specific, more obviously grounded in Elizabethan social relations,† (63) thus the theme of misrule is represented in a more literal fashion. 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Review Of Higher Learning - 966 Words

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