“DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIRVALIDATION

FOR THE DETERMINATION OF ETODOLAC AND ANALGIN IN BULK AND MARKETED FORMULATIONS”

MASTER OF PHARMACY

DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTHSCIENCES, KARNATAKA,

BANGALORE

BY

BHALANI MONABEN CHIMANBHAI

Under The Guidance of

Dr.E.V.S.Subrahmanyam.M.PHARM.Ph.D

P.G. DEPARTMENT OF QUALITY ASSURANCE,

SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143

2013-2015

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE –II

REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 / NAME AND ADDRESSOF
THE CANDIDATE / BHALANI MONABEN CHIMANBHAI
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL,POST PARENGIPETE,
MANGALORE TQ-574143
2.0 / NAME OF THE
INSTITUTION / SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE.
3.0 / COURSE OF STUDY &
SUBJECT / MASTER OF PHARMACY
(QUALITY ASSURANCE)
4.0 / DATE OF ADMISSION / 25th JULY 2013
5.0 / TITLE OF THE TOPIC:
“DEVELOPMENT OF NEW ANALYTICAL METHODS ANDTHEIRVALIDATION FOR THEDETERMINATION OF ETODOLAC AND ANALGININ BULK AND MARKETED FORMULATIONS”
6.0
7.0
8.0 / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for study:

Analytical Method Development for Pharmaceutical Formulations:

Analytical methods are essential to characterize drug substances and drug products composition during all stages of pharmaceutical development. For routine analytical purpose it is always necessary to establish methods capable of analyzing large number of samples in a short time period with high accuracy and precision.
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 new analytical methods for such drugs. These products can present challenges to the analytical chemist responsible for the development andvalidation of analytical methods.
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.
Method development scheme for a typical HPLC-UV related substance describe below,
  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 an appropriate standardization method and the use of relative response factors in calculations.
  5. To identify the “weaknesses” 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).
6.3 A BRIEF INTRODUCTION ABOUT ETODOLAC1,2:
Chemical structure of Etodolac:

Chemical name: (RS)-2-(1,8-Diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-
yl)acetic acid
Empirical formula:C17H21NO3
Molecular weight:287.35
Solubility: Methanol and Ethanol
Protein binding:100%
Half-life: 7.3±4.0 hours
Excretion:renal
Melting point:145-148ºC
Characteristics: White crystalline compound
PHARMACOLOGY1,2:
DRUG CATEGORY:
  • Anti-inflammatory Agents
  • Anti-rheumatic Agents

MECHANISM OF ACTION:
NSAIDs are used for the management of mild to moderate pain, fever, and inflammation. They work by reducing the levels of prostaglandins, which are chemicals that are responsible for pain and the fever and tenderness that occur with inflammation. Etodolacblocks the enzyme that makes prostaglandins (cyclooxygenase), resulting in lower concentrations of prostaglandins. As a consequence, inflammation, pain and fever are reduced.
Post-marketing studies demonstrated that etodolac inhibition of cyclooxygenase is somewhat COX-2 selective [2] similar to celecoxib and other "COX-2 inhibitors." Unlike rofecoxib, both etodolac and celecoxib can fully inhibit COX-1 and are designated as having "preferential selectivity" toward COX-2. The (inactive against COX) r-enantiomer of etodolac inhibits beta-catenin levels in hepatoma cells.

SIDE EFFECTS:

Etodolac may cause side effects, or adverse drug reactions. It is advised to contact a physician if any of these symptoms are severe or persistent: Constipation, diarrhoea, gas or bloating, vomiting, headache, dizziness, ringing in the ears, running nose, sore throat and blurred vision.
Some side effects can be serious. It is advised if any of the following symptoms occur to immediately contact a physician, and to discontinue use until such a time.
Unexplained weight gain, swelling of the eyes, face, lips, tongue, throat, hands, feet, ankles, or lower legs, fever or chills, blisters, rash, itching, hives, hoarseness, difficulty breathing or swallowing, yellowing of the skin or eyes, excessive tiredness, unusual bleeding or bruising, lack of energy, loss of appetite, pain in the upper right part of the stomach, flu-like symptoms, pale skin, fast heartbeat, cloudy, discolored, or bloody urine, difficult or painful urination, back pain.
6.4 REVIEW OF LITERATURE:
A great deal of work has been done by the scientist about the current application and future possibilities for altering the drug activities and evaluation with new method development by instrumental methods.
  1. Ayman A Gouda and Wafaa S Hassan3have reportedSpectrophotometric determination of Etodolac in pure form and pharmaceutical formulations. The determination was based on the oxidation of etodolac by Fe3+ in the presence of o-phenanthroline(o-phen) or bipyridyl(bipy) and/or Fe+3-bipy mixtures in acetate buffer solution at optimum pH at 510 & 520 nm with o-phen&bipy. Standard deviations were ≤ 0.76% with recoveries of 99.87% - 100.21%.
  2. Mit J. Patel, R. Badmanaban and C. N. Patel4have reported A reversed-phase liquid chromatographic (RP-HPLC) method was developed for the simultaneous determination of tolperisone hydrochloride (TOLP) and etodolac (ETD) in a combined fixed dose oral formulation. The analysis was carried out using a phenomenax C-18, pre-packed column. A mobile phase containing a phosphate buffer (pH 5.5) : Methanol : Acetonitrile : Tri-ethylamine (40 : 40 : 20 : 1.5), with the pH adjusted to orthophosphoric acid, was pumped at a flow rate of 1.0 ml min1 with a UV-detector and PDA detection at 257 nm. Retention time was 3.91 minutes and 6.89 minutes for TOLP and ETD, respectively. The method was validated for linearity, accuracy, precision, sensitivity, and specificity. The method showed good linearity in the range of 3 – 21 μg ml for TOLP μg / ml and 8 – 56 μg / ml for ETD. The detection limit of the proposed method was 0.16 μg / ml and 0.58 μg / ml for TOLP and ETD, respectively. The quantification limit of the proposed method was 0.51 μg / ml and 1.7 μg / ml for TOLP and ETD, respectively. The % recovery was within the range of 99.42 – 101.15 for TOLP and 98.63 – 100.94 for ETD. The percentage RSD for precision of the method was found to be less than 2%. The method was validated as per the International Conference on Harmonization (ICH) guidelines. The developed method could be applied for routine analysis of TOLP and ETD in tablet dosage form.
  3. Vaijanath G. Dongre, Sweta B. Shah, Gunaji S. Bayer, ManishaPhadke,Vivek K. Jadhav5have reportedSimultaneous Determination of Etodolac and Acetaminophen in Tablet Dosage Form by RP-LC. A simple, specific, precise and accurate reverse phase liquid chromatographic (RP-LC) method has been developed for the simultaneous determination of etodolac and acetaminophen in tablet dosage form. The chromatographic separation was achieved on a BDS Hypersil C18, 100 mm × 4.6 mm, 5 μm column at a detector wavelength of 274 nm using an isocratic mobile phase consisting of a mixture of 0.05% aqueous orthophosphoric acid and acetonitrile in the ratio of 50:50 (v/v) at a flow rate of 1.0 mL min−1. The retention times for etodolac and acetaminophen were found to be 1.32 and 4.24 min, respectively. The method was validated for the parameters like specificity, linearity, precision, accuracy and robustness. The method was found to be specific and stability indicating as no interfering peaks of impurities, degradant and excipients were observed. The square of correlation coefficients (R 2) for etodolac and acetaminophen were 0.9996 and 0.9998 while percentage recoveries were 101.32 and 100.94%, respectively. Intra- and inter-day relative standard deviations for both the components were <2.0%. The proposed RP-LC method can be applied for the routine analysis of commercially available formulations of these drugs either as such or in combination.

4.JadhavAlpa V., Gohel A., Bhavika.,SondagarMital M., Patel Bhavna A., ParmarShraddha J.6 have reported Method Development And Validation For The Simultaneous Estimation Of Paracetamol And Etodolac By Derivative UV Spectroscopic Method. A simple, novel, sensitive, precise and specific validated Spectrophotometric method was developed for simultaneous determination of Paracetamol and Etodolac in synthetic mixture and its dosage form. Methanol: Water (60:40) was selected as a common solvent for estimation of Paracetamol and Etodolac with λmax at 247 nm and 280 nm respectively in methanol: water (60:40 v/v). Derivative method was selected for the estimation of both the drug simultaneously. The linearity was obtained in the concentration ranges of 5-25 µg/ml for Paracetamol and 2-18 µg/ml for Etodolac. The Zero Crossing Point (ZCP) of Paracetamol was 219.27 nm and Etodolac was 224.28 nm. The correlation coefficient was found to be 0.9994 and 0.9983 for Paracetamol and Etodolac respectively. The detection limit and quantification limit were found to be 0.34 and 1.02 µg/ml for Paracetamol and 0.37 and 1.11 µg/ml for Etodolac respectively. The method was validated as per the International Conference on Harmonization (ICH) guidelines.

5.Thankappan Surya; Parmar Ashok; Sailor Bhavika; VekariyaKinjal; Khasia Vasant7have reportedDevelopment and Validation of Spectroscopic method for Simultaneous Estimation of Etodolac and Thiocolchicoside in tablet formulation. Simple, precise and economical spectrophotometric method has been developed for simultaneous estimation of Etodolac and Thiocolchicoside in combined tablet dosage form. The first method is based on the use of simultaneous equation method(Method A) and the second method is based on Absorbance correction method(Method B). In method A, absorbance is measured at two wavelengths, one being λmax of etodolac 223nm and the other being λmax of thiocholchicoside at 260nm. In method B, one wavelength is the λmax of etodolac 223nm and other wavelength is 360nm(where ETD does not interfere in absorbance of TCD). Both the drugs obey the Beer's law in the concentration ranges employed for these methods. The methods were validated by following the analytical performance parameters suggested by International Conference on Harmonization. All validation parameters were within the acceptable range. These developed methods can be applied for routine analysis.

  1. Rohit Shah, ChandrakantMagdum, ShitalkumarPatil, Dhanya Kumar Chougule and Nilofar Naikwade8havereportedValidated Spectroscopic Method for Estimation of Aceclofenac from Tablet Formulation. Aceclofenac is a non steroidal anti-inflammatory drug with good analgesic and anti-rheumatic properties. Various methods for analysis of the same are available but are time consuming and expensive. Here they have developed a new, precise and simple UV spectrophotometric method for estimation of aceclofenac from tablet formulation. The drug obeyed the Beer’s law and showed good correlation. It showed absorption maxima at 273 nm; in phosphate buffer pH 7.4. The linearity was observed between 0 – 20 mcg/mL. The results of analysis were validated by recovery studies. The recovery was more than 99%. The method was found to be simple, accurate, precise, economical and robust.
6.5A BRIEF INTRODUCTION OF ANALGIN9:
Chemical structure ofAnalgin:

Iupac name: sodium [(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-
pyrazol-4-yl)methylamino] methanesulfonate
Synonyms: Metimazole, Novalgin, Dipyrone
Formula:C13H16N3O4S.Na
Molecular weight: 333.337
Solubility : Water and Ethanol
Bioavailability: 85%
Protein binding: 58-60%
Half-life:1-4 hours
Excretion: renal
Melting point :222-226°C
Characteristics: A white or yellowish crystalline powder.
PHARMACOLOGY10,11:
MEDICAL USES:
Analgin is used for the treatment of pains of different origin and variable intensity: toothache, headache, arthralgia, neuralgia, myositis, mild to moderate visceral pain, high fever, not responding to other drugs.
MECHANISM OF ACTION:
The exact mechanism and location of action of metamizole are not explained in detail. It is assumed that it operates in a combined central and peripheral effects. The central action is explained by inhibition of prostaglandin synthesis, secretion of histamine and serotonin from mast cells of thalamus. Peripheral effect is explained by the inhibition of prostaglandin synthesis which sensitize nociceptors to the effect of algogen mediators. In addition, metamizolehas a direct blocking effect on the inflammatory hyperalgesia.Analgin is a medicine of the pyrazolone group, possessing hard analgesic and antipyretic effects and moderate antiinflammatory activity. Blocking of the synthesis of endogenous pyrogens - prostaglandins D and E - is the cause for the antipyretic activity and also for the analgesic action of this drug. The decrease of the prostaglandins production in the periphery (and respective decrease of nerve endings sensitivity) plays a relatively smaller role. In contrast to the other nonnarcotic analgesic drugs, Analgin stimulates the release of β-endorphins, explaining its activity in cases of visceral pain. Analgin has a slight spasmolytic activity on the smooth muscle cells of the biliary and urinary tracts and also on the muscle of the uterus.
SIDE EFFECTS:
The serious adverse reactions after administration might include a shock. Signs of the starting shock are cold sweat, dizziness, nausea, skin discoloration, shallow breathing. It is accompanied by precordial tightness, rapid pulse, cold extremities and a sharp drop in blood pressure. Immediately after the onset of first symptoms the administration should be discontinued and the intense shock therapy must be applied.
Another serious side effect is blood dyscrasias (agranulocytosis, leukopenia, thrombocytopenia).
Uncommon side effects: allergic rash, low blood pressure.
Rare side effects: spotty pustel-rashes, white-blood cell-deficiency.
Very rare side effects: Schmerzmittel-asthma, severe skin reactions (Stevens-Johnson Syndrome, Lyell Syndrome), circulatory shock, lack of granulocytes, lack of platelets, deterioration of kidney function, protein in the urine, urinary excretion deficiencies, Harnsperre, inflammation of the blood vessels (for injection), emotional disorders (anxiety, agitation, delirium, depression, delusions, apathy).
Specialties: Vereinzelt particularly serious cases of skin reactions can occur such as the Toxic Epidermal Necrolysis (TEN). Called this also staphylogenes Lyell Syndrome cutaneous reaction leads to the large blasigen skin separation and is a severe, life-threatening disease.
During the application of Analgin there may be agranulocytosis and lack of platelets to a life-threatening blood-formation disturbance, therefore the blood count should be controlled regularly by doctors. The patient gets fever after application of the active substance or he noticed subcutaneous bleeding, immediately discontinue treatment and the doctor is to ask.
When injections, pain in the injection site and local reactions at the injection site may occur in some cases. Also, there can be a strong drop in blood pressure in an injection into the veins in rare cases.
6.6REVIEW OF LITERATURE:

1.Dr. K. Raghubabu and B. Kalyana Ramu12have reported Development of New Visible Spectrophotometric Determination of Analgin in Bulk & Formulations using p-Anisidine –ferric chloride as Oxidative Coupling Reagent. A simple and sensitive visible spectrophotometric method has been developed for the determination of analgin from its bulk drug and formulations. This method is based on the oxidative coupling reaction with p-anisidine in the presence of iron (III) to yield the violet-red colored product having maximum absorbance at 550nm. Regression analysis of Beer's law plot showed good correlation in the concentration range of 4.0-40.0μg/ml. The proposed method is applied to commercial available tablets and the results are statistically compared with those obtained by official method and validated by recovery studies. The results are found satisfactory and reproducible. The method is applied successfully for the estimation of the analgin in formulations without the interference of excipients. The method offers the advantages of rapidity, simplicity and sensitivity, normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

2.V. Vlasova, A. V. Shilova and Yu. S. Fokina13have reported Spectrophotometric Assay of Active Components in Medicinal Formulations Using the Vierordt Method for Analysis of Analgin-quinine and Panadol Extra. A method for the spectrophotometric assay of analgin, caffeine, quinine hydrochloride and paracetamol in two-component medicinal formulations was developed using a new criterion for selecting analytical wavelengths (AWL). Content calculations were performed using several sets of AWL. Model solutions were used to show that use of three sets of AWL decreases estimation errors for components in mixtures withboth low and high content. This method was used to analyze the medicinal formulations Panadol Extra and Analgine-quinine.

3.Saidul Zafar Qureshi, Ahsan Saeed, Tausiful Hasan14have reportedSpectrophotometric Determination of Novalgin in tablets by use of Potassium Iodate.An indirect colour reaction has been studied for determination of novalgin in tablets. The method is simple, rapid and reproducible with a relative standard deviation of 0.2%. Novalgin is determined spectrophotometrically by means of its colour reaction with potassium iodate. Beer's law is obeyed over the range 1–10 mg of drug. A tentative reaction mechanism has been proposed.

4.Hamide Z. Senyuva, SureyyaOzcan,BurakVeli Kabasakals15have reported Validated Simple, Rapid and Accurate HPLC Methods for determination of Metamizole Sodium in Solid and Liquid Dosage Forms. The aim of the study was to develop and validate high performance liquid chromatography (HPLC) assay for the rapid determination of Metamizole Sodium in solid and liquid dosage forms. The experimental procedure involved reversed-phase- HPLC with a Zorbax SB C18 column (5 µm particle size, 4.6 ID x 250 mm), metanol- water volumetric solution (80: 20, v/v) mobile phase, UV detection at 254 nm for Metamizole Sodium. The flow rate of the, mobile phase was 1 mL/min. The retention time of Metamizole Sodium is ca. 3.1-3.3 min. Formulations components did not give rise to any interfering peaks. Calibration curve was linear over the range 0.5-100 µg/mL for analyte.Themetamizole recovery range is at three level (10µg/mL, 15µg/mL and 25µg/mL) 93-100 %. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both solid and liquid formulations.