Rajiv Gandhi University of Health Sciences s146

“DEVELOPMENT OF NEW ANALYTICAL METHOD AND ITS VALIDATION FOR THE DETERMINATION OF ETHAMSYLATE IN BULK AND MARKETED FORMULATIONS”

MASTER OF PHARMACY DISSERTATION PROTOCOL, SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

BY BHOJANI ANILKUMAR RAMESHBHAI M.PHARM – I

Under The Guidance Of

Mrs. Padmavathi. P. Prabhu M.PHARM.

DEPARTMENT OF QUALITY ASSURANCE.

SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143. 2011 – 2013

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE – ІІ

REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 NAME OF THE CANDIDATE BHOJANI ANILKUMAR RAMESHBHAI, ADDRESS SRINIVAS COLLEGE OF PHARMACY, VALACHIL,POST PARENGIPITTE, MANGALORE-574143

2.0 NAME OF THE INSTITUTION SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE.

3.0 COURSE OF STUDY & MASTER OF PHARMACY SUBJECT DEPARTMENT OF QUALITY ASSURANCE

4.0 DATE OF ADMISSION 15th JUNE 2011

5.0 TITLE OF THE TOPIC:

“DEVELOPMENT OF NEW ANALYTICAL METHOD AND ITS VALIDATION FOR DETERMINATION OF ETHAMSYLATE IN BULK AND MARKETED FORMULATIONS”

2 6.0 BRIEF RESUME OF THE INTENDED WORK:

6.1 NEED FOR STUDY:

 Analytical Method Development for bulk drug and Pharmaceutical Formulations: The number of drugs, which may be either new entities or partial structural modification of the existing ones, introduced into the market is increasing every year. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. Hence, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs.

6.2 Basic criteria for new method development of drug analysis:

 The drug or drug combination may not be official in any pharmacopoeias.  A proper analytical procedure for the drug may not be available in the literature due to patent regulations.  Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.  Analytical methods for a drug in combination with other drugs may not be available.  The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.

Analytical method development provides the support to track the quality of the product from batch to batch. Estimation can be performed by the following two methods:  Titrimetric methods and  Instrumental methods. . Spectrophotometric Methods . Chromatographic Methods

Methods for analyzing drugs in dosage forms can be developed, provided one has knowledge about the nature of the sample, its molecular weight, polarity, ionic character and the solubility parameter. Method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase and what type of reagent is use. The following is a suggested method development scheme for a typical HPLC-UV related substance method.

1. To define the goals for method development (e.g., what is the intended use of the method?), and to understand the chemistry of the analytes and the drug product.

2. To develop preliminary HPLC conditions to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments.

3. To develop a suitable sample preparation scheme for the drug product

4. To determine the appropriate standardization method and the use of relative response factors in calculations.

5. To identify the drawbacks of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples.

6. To complete method validation according to ICH guidelines as mentioned in Q2 (R1). Single dosage forms with combination of drugs are widely used today due to their advantages and their simultaneous estimation of individual component is a challenging task.

4 1 6.3 GENERAL DISCUSSION ON ETHAMSYLATE :

Ethamsylate (brand names: E-SYLATE-250, Ethamcip-250) is haemostatic drug.It is believed to work by increasing capillary endothelial resistance and promoting platelet adhesion. It also inhibits biosynthesis and action of those prostaglandins which cause platelet disaggregation, vasodilation and increased capillary permeability

STRUCTURE :-

IUPAC Name: - 2, 5 – dihydroxybenzene sulfonic acid; N – ethylethanamine.

Formula :- C10H17NO5 S

Mol. Weight: - 263.31068 g/mol.

Drug category: - Hemostatic agent. Physical state: - White crystalline powder.

Solubility: - Easily soluble in water, soluble in ethanol and slightly soluble in acetone, insoluble in chloroform and ether.

Mechanism of action:-Ethamsylate is a haemostatic agent; also promotes angioprotective and proaggregant action. It stimulates thrombopoiesis and their release from bone marrow. Haemostatic action is due to activation of thromboplastin formation on damaged sites of small blood vessels and decrease of PgI2 (Prostacyclin I2) synthesis; it also facilitates platelet aggregation and adhesion, that at last induce decrease and stop of hemorrhage. The precise mechanism of action of Ethamsylate is unknown. It has been shown to reduce bleeding time and blood loss from wounds. This appears to relate to increased platelet aggregation mediated by a thromboxane A2 or prostaglandin F2a dependent

5 mechanism. It has also been associated with decreased concentrations of 6- oxoprostaglandin F1a, a stable metabolite of prostacyclin. Prostacyclin is a potent vasodilator, and may be implicated in reperfusion; it is also a disaggregator of platelets. Whereas prostaglandins themselves may have a role in regulating cerebral blood flow, Ethamsylate appears to have no effect on cerebral blood flow. Ethamsylate was also thought to stabilise capillaries, reinforcing capillary membranes by polymerising hyaluronic acid. Ethamsylate limits capillary bleeding through its action on hyaluronic acid and initial studies showed a reduction in intraventricular haemorrhage. Ethamsylate may also have an effect on the microcirculation, encouraging platelet aggregation and vasoconstriction and therefore haemostasis. It also inhibits the effects of the prostaglandin mediated vasodilatation and increased capillary permeability, thereby reducing oedema secondary to capillary leakage. It is also possible that Ethamsylate would reduce reperfusion haemorrhage in ischaemic areas of the brain, preventing secondary damage. By inhibiting the effects of prostaglandins, Ethamsylate may exert an effect by closing the patent ductus and thereby increasing cerebral blood flow.

6.4 REVIEW OF LITERATURE :- A literature survey was carried out for the estimation of Ethamsylate in marketed dosage forms. It was found that a few methods have been reported for these drugs. The collection of references are reproduced below

 Nagaraju P, Krishnachaithanya K, Srinivas VDN and Padma SVN.2 have developed Validated RP-HPLC Method for the Determination of Ethamsylate in Bulk and Tablet Dosage Forms. The HPLC separation was carried out by reverse phase chromatography on C18 column (4.6 x 250 mm, 5μm particle size) with a mobile phase composed of methanol: acetonitrile: acetate buffer PH 2.8 (60:30:10 v/v) in isocratic mode at a flow rate of 1 ml / min. The detection was monitored at 290 nm. The calibration curve for Ethamsylate was linear from 0.5 – 100 μg/ml. The interday and intraday precision (relative standard deviation) was found to be within the limits.

6  Vamshikrishna N, Shetty ASK.3 have developed Validated RP-HPLC Method for the Estimation of Ethamsylate in Bulk drug and Pharmaceutical formulations. Ethamsylate was chromatographed on a reverse phase C18column (25cm x 4.6 mm i.d; particle size 5 μm) in a mobile phase consisting of methanol and disodiumhydrogen orthophosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 0.6 ml/min with detection at 290 nm. The detector response was linear in the concentration of 10-50 μg/ml. The limit of detection and limit of quantitation was found to be 0.89 and 2.7 ng/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.83%.

 Roshan I, Kaushik KV, Diptish KN.4 have developed a spectrophotometric method for simultaneous estimation of Ethamsylste and Tranexamic acid in combined tablet dosage form. This method employs derivatization procedure for making tranexamic acid UV detectable. It employs formation and solving of simultaneous equation using two wavelengths 299.0 nm and 286.2 nm. This method obeys Beer’s law in the employed concentration ranges of 4-15 μg mL-1 and 2-12 μg mL-1 Ethamsylate and Tranexamic acid respectively. Results of analysis were validated statistically and by recovery studies.

 Arshad MD, Arvind BK, Venugopal RD, Sajja AR.5 have developed two simple and economical validated visible spectrophotometric methods (A and B) for the Estimation of Ethamsylate in bulk drug and pharmaceutical dosage forms. Method A is based on the reaction of Ethamsylate with Folin-Ciocalteu(phenol’s) reagent under alkaline conditions forming a blue colored chromogen exhibits absorption maxima at 619 nm. Beer’s law was obeyed in the concentration range of 10-50 µg/ml. In Method B Ethamsylate undergoes oxidation followed by complex formation reaction with 1, 10 phenanthroline in presence of ferric chloride to form red colored chromogen exhibiting absorption maxima at 510 nm and obeyed Beer’s law in the concentration range of 1-5 µg/ml.

7  Vinay KB, Revanasiddappa HD, Okram ZD, Basavaiha K.6 have developed two spectrophotometric methods for the determination of Ethamsylate(ETM) in bulk drug and in tablets employing permanganate as the oxidimetric reagent. A direct spectophotometry (Method-A) involves treating the aqueous solution of the drug with permanganate in alkaline medium and measuring the bluish green product at 610 nm. In indirect spectrophotometry (method B), the drug solution was treated

with a fixed concentration of permanganate in H2SO4 medium, and after a specified time, the unreacted permanganate was measured at 545 nm. In spectrophotometry, Beer’s law is obeyed over 0.5- 5.0 and 1.5– –15 μg ml-1 for method A and B, respectively.

 Vinay KB, Revanasiddappa HD, Okram ZD, Basavaiha K.7 have developed two spectrophotometric methods for the determination of Etamsylate(ETM) in bulk and capsule formulations using ferric chloride based on complex formation reactions. The methods are based on the oxidation of ETM with ferric chloride in neutral medium and subsequent chelation of the resulting iron (II) with 1, 10- phenanthroline (Phen) (method A) and with 2, 2’-bipyridyl (Bipy) (method B). The resulting red colored chromogens are measured at 510 and 520 nm, in method A and B, respectively. Beer’s law is obeyed over the ranges 0.5-10 and 0.8-16 μg/ml for method A and B, respectively.

 Neeraj K, Himani A, Abhijit K, Dhaneshwar SR.8 have developed two sensitive and reproducible methods are described for the quantitative determination of Ethamsylate in the presence of its degradation products. The first method was based on high-performance liquid chromatographic (LC) separation of the drug

from its degradation products on the reversed phase, kromasil column [C18 (5-μm, 25 cm × 4.6 mm, i.d.)] at ambient temperature using a mobile phase consisting of methanol and water (50:50, v/v). Flow rate was 0.6 ml min−1 with an average

−2 operating pressure of 180 kg cm and retention (tR) time was found to be 2.93 ± 0.05 min. The second method was based on high-performance thin layer chromatographic (HPTLC). The separation was carried out on Merck HPTLC 8 aluminium sheets of silica gel 60 F254 using toluene:methanol:chloroform (8.0:4.5:6.0, v/v/v) as mobile phase. . This system was found to give compact

spots for Ethamsylate after double development (retention factor, Rf value of 0.23 ± 0.02). The second order polynomial regression analysis data was used for the regression line in the range of 500–6000 ng spot−1.

 Yogini SJ, Gokul ST, Sanjay JS.9 have developed HPTLC method for determination of Ethamsylate and Mefenamic acid in tablets. Identification and

quantification were performed on silica gel 60 F 254 HPTLC plates, prewashed with methanol, with chloroform-methanol-acetic acid (10:8:0.2 v/v) as mobile phase. The validated calibration ranges were 500–2500 ng spot −1 (r = 0.998) and 500–2500 ng spot −1 (r = 0.997) for Ethamsylate and Mefenamic acid, respectively. The spots were scanned at λ = 300 nm.

 Fathalla B, Amina El-B, Nahed El, Manar T.10 have developed two spectrofluorometric methods were developed for determination of Ethamsylate(ETM). Method I is based on measuring the native fluorescence of Ethamsylate in water at 354 nm after excitation at 302 nm. The calibration plot was rectilinear over the range of 0.05–1 μg/mL for ETM with limits of detection and quantitation of 7.9 and 26ng/ml, respectively. Method II involved synchronous and first derivative synchronous fluorimetric methods for the simultaneous determination of Ethamsylate (ETM) and hydroquinone (HQ). The synchronous fluorescence of both the drug and its impurity were measured in methanol at Δ λ of 40 nm. The peak amplitudes (1D) were estimated at 293.85 or 334.17 nm for ETM and at 309.05 nm for HQ. Good linearity was obtained for ETM over the ranges 0.1–1.4 μg/mL and 0.1–1.0 μg/mL at 293.85 and 334.17 nm, respectively. For HQ, the calibration plot was rectilinear over the range of 0.01– 0.14 μg/mL at 309.05 nm. Limits of detection were 20, 2.01 ng/mL and limits of quantitation were 60, 6.7 ng/mL for ETM and HQ by method II, respectively.

6.5 OBJECTIVES OF THE STUDY:-

9 In the proposed work, attempt shall be made :

 To develop a new method for the estimation of Ethamsylate.

 To develop a validated method according to ICH guidelines.

 To apply validated method for the estimation of Ethamsylate in pharmaceutical formulation.

7.1 MATERIALS AND METHODS:-

 Drug: Ethamsylate. 7.0  Reagent: Ethanol, Acetone, Bromothymol blue, 4 – aminophenazine, P - dimethylamino benzaldehyde -orthophosphoric acid.

METHOD DEVELOPMENT:-

 All experiments shall be carried out in the Department of Quality Assurance. Srinivas college of Pharmacy, Mangalore.  Pure samples of Ethamsylate shall be procured from Industries involved in bulk manufacture of this drug.  Dosage formulations shall be procured from local market.  UV spectrophotometer Shimadzu-UV1700 with spectral band width of 2nm and 10nm and matched quartz shall be used for measuring absorbance of drug solutions.  HPLC instrument JASCO ISOCRATIC HPLC-2000 SYSTEM with C18 column shall be used.

7.2 SOURCE OF DATA:-

 References from library – Srinivas college of pharmacy  www.pharmainfo.net.  www.google.com  www.sciencedirect.com

10  www.rxlist.com  www.medline.com

7.3 Does the study require any investigation to be conducted on patients or animals?

Not applicable

7.4 Has the ethical clearance been obtained from your institution in case of 7.3? Not applicable

REFERENCE: 1. www.Rxlist.com. 2. Nagaraju P, Krishnachaithanya K, Srinivas VDN, Padma SVN. Reverse Phase 8.0 HPLC Method for the Determination of Ethamsylate in Bulk and Tablet Dosage Forms. Asian J. Research Chem 2008; 1(2):88-90. 3. Vamshikrishna N, Shetty ASK. Development and Validation of RP-HPLC Method for the estimation of Ethamsylate in Bulk drug and Pharmaceutical formulations. Int.J. ChemTech Res 2011; 3(2):928-32. 4. Roshan I, Kaushik KV, Diptish KN. Spectrophotometric Methods for Simultaneous Estimation of Ethamsylate and Tranexamic Acid from Combined Tblet dosage Form. Int.J. ChemTech Res. 2010; 2(1):74-8. 5. Arshad MD, Arvind BK, Venugopal RD, Sajja AR. Development and Validation of Visible Spectrophotometric methods for the Estimation of Ethamsylate in Pharmaceutical dosage Forms. Der Pharma Chemica 2011; 3(2):347-51. 6. Vinay KB, Revanasiddappa HD, Okram ZD, Basavaiah K. Permanganometric Determination of Ethamsylate in Bulk Drug and in Tablets. CI&CEQ 2009; 15(3):149-57.

11 7. Vinay KB, Revanasiddappa HD, Okram ZD, Basavaiah K. Spectrophotometric Determination of Ethamsylate in Pharmaceuticals using Ferric Chloride based on Complex Formation Reactions. CI&CEQ 2010; 16(1):1-9. 8. Neeraj K, Himani A, Abhijit K, Dhaneshwar SR, Bharat P. Stress degradation amstudies on etamsylate using stability-indicating chromatographic methods. Analytica Chimica Acta 2005; 536(1-2):49-70. 9. Yogini SJ, Gokul ST, Sanjay JS. Quantitative Analysis of Ethamsylate And Mefenamic Acid in Tablets by use of Planar Chromatography. Journal of Planar Chromatography 2005; 18:460-64. 10. Fathalla B, Amina El-B, Naheb El-E, Manar T. Conventional and First Derivative Synchronous Fluorometric Determination of Ethamsylate in Pharmaceutical Preparation and Biological Fluids. Journal of Fluorescence 2011; 21(4):1371-84.

9 SIGNATURE OF THE CANDIDATE (Bhojani Anilkumar.R.)

12 10 REMARKS OF THE GUIDE Forwarded for Approval Mrs.Padamavathi P. Prabhu , NAME AND DESIGNATION OF 11 Department of Q.A. GUIDE Srinivas College of Pharmacy.

11.1 SIGNATURE

12 CO-GUIDE

12.1 SIGNATURE Dr. E.V.S Subrahmanyam, Profesor and head, 13 HEAD OF THE DEPARTMENT Department of Q.A. Srinivas College of Pharmacy.

13.1 SIGNATURE

14 REMARKS OF THE PRINCIPAL

Forwarded for approval

Dr. Ramakrishna Shabaraya A., 15 SIGNATURE OF PRINCIPAL Principal, Srinavas College of Pharmacy, Valachil,Mangalore.