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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

|1 |NAME AND ADDRESS OF THE CANDIDATE |PRIYANKA SEEGARAGADDA, |

| | |D/O S.Sivakanth, |

| | |H.NO:5-1/42,Mythrinagar Phase-3, |

| | |Madinaguda , near Miyapur, |

| | |Hyderabad-500050 |

| | |Andhra Pradesh |

| | | |

| | |POSTAL ADDRESS |

| | |KRUPANIDHI COLLEGE OF PHARMACY |

| | |Chikkabellandur,Carmelaram Post |

| | |Varthur Hobli, Sarjapur Road |

| | |Bangalore 560035 |

| | |Karnataka |

|2 |NAME OF THE INSTITUTUE |KRUPANIDHI COLLEGE OF PHARMACY |

| | |Chikkabellandur |

| | |Carmelaram Post |

| | |Varthur Hobli, Sarjapur Road |

| | |Bangalore 560035 |

| | |Karnataka |

|3 |COURSE OF THE STUDY AND SUBJECT |Master of Pharmacy (Quality Assurance) |

|4 |DATE OF THE ADMISSION OF THE COURSE | 16-11-2011 |

|5 |TITLE OF THE PROJECT |

| | |

| |Analytical Method Development and Validation for a Novel Ophthalmic Formulation by RP-HPLC |

|6 |BRIEF RESUME OF THE INTENDED WORK |

| | |

| |6.1 NEED OF THE STUDY |

| | |

| |The concept of analytical chemistry lies in the simple, precise and accurate measurements. These determinations require |

| |highly sophisticated instruments and methods like mass spectrometry, gas chromatography, HPTLC, HPLC, etc. HPLC method is |

| |sensitive, accurate, precise and desirable for routine estimation of drugs in formulations. Thereby it is advantageous |

| |than volumetric methods. Many HPLC methods have been developed and validated for the quantitative determination of various|

| |marketed drugs.1 |

| |Analytical method development and validation plays an important role in drug discovery and manufacture of pharmaceuticals.|

| |These methods are used to ensure the identity, purity, potency and performance of drug products majority of analytical |

| |development effort goes into validating a stability indicating method. So it is a quantitative analytical method based on |

| |the structure and chemical properties of each active ingredient of the drug formulation. |

| |Most of the drugs can be analyzed by HPLC method because of several advantages like rapidity, specificity, accuracy, |

| |precision reproducibility, ease of automation and eliminates tedious extraction and isolation procedures. |

| |Hence the need of the study is to carry out the method development and validation for quantitative estimation of |

| |ophthalmic solutions in bulk drug and pharmaceutical dosage form by HPLC as per ICH guidelines. |

| | |

| |6.2 REVIEW OF LITERATURE |

| | |

| |Vidal-ollivier E has determined the ophthalmic drug , Guiazulene by HPLC.2 |

| | |

| |Vidal E has determined ophthalmic therapeutics of Metipranolol and its degradation product by RP-HPLC.3 |

| | |

| |Alireza S. Kord has stated that a novel HPLC method for determination of EDTA in a cataract inhibiting ophthalmic drug |

| |product has been developed. In this method EDTA was converted to Cu(II)EDTA complex, using Cu+2 containing mobile phase, |

| |after injection into the chromatographic system. This allowed complexation of EDTA with Cu+2 without interference from |

| |formulation ingredients. The Cu(II)EDTA complex was separated from drug substance, impurities, degradants and other |

| |formulation excipients by a 250 × 4.1 mm anion exchange column and detected at UV wavelength 250 nm. The mobile phase |

| |consisted of 2 mM cupric nitrate, 11 mM nitric acid, and 25% (v/v) acetonitrile at pH 3.0. This stability indicating assay|

| |has been validated and shown to be specific, linear, precise, accurate and rugged for routine EDTA analysis.4 |

| | |

| |Randolph D. Glickman stated that four different antibiotics, delivered individually to rabbit eyes via hydrophilic |

| |intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples |

| |from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) |

| |method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. |

| |Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were |

| |eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM |

| |tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH |

| |5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds |

| |approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to |

| |200 pg. The overall accuracy of the method was ≥90%. With minor modifications, the method was optimized for each of the |

| |agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular |

| |penetration of these drugs.5 |

| | |

| |Alain Carrier has stated that a simple and rapid reversed-phase HPLC method was developed for routine analysis of total |

| |benzalkonium chloride in ophthalmic formulations. The analysis involves simple sample preparation using the mobile phase |

| |as the diluent. The method uses a Waters SymmetryShield RP-18 (75 mm × 4.6 mm, 3.5 μm particle size) column and a mobile |

| |phase consisting of a mixture of methanol–potassium phosphate (pH 3.0; 7.5 mM) (68:32, v/v). Using these conditions, three|

| |major homologs of the benzalkonium chloride (C12, C14 and C16) were separated in less then 7 min. Furthermore, recoveries |

| |ranging from 97% to 99% at three levels of the label claim of total benzalkonium chloride content were obtained for |

| |different ophthalmic formulations. Data supporting the development and validation of this method are presented.6 |

| | |

| |Gurny R stated that quantification of pilocarpine in the presence of the epimer isopilocarpine in polymeric dispersions is|

| |reported. The method describes a technique using reversed-phase HPLC and UV detection of pilocarpine in the presence of a |

| |pH-sensitive polymer. As this method does not require prior sample preparation it will be of special interest for process |

| |control and development.7 |

| | |

| |Nageswara Rao R stated that an extensive survey of the literature published in various analytical and pharmaceutical |

| |chemistry related journals has been conducted and the high-performance liquid chromatography (HPLC) methods which were |

| |developed and used for determination of process-related impurities in drugs have been reviewed. This review covers the |

| |time period from 1995 to 2001 during which around 450 analytical methods including all types of chromatographic and |

| |hyphenated techniques were reported. HPLC with UV detection was found to be the technique of choice for many workers and |

| |more than 200 methods were developed using LC-UV alone. A critical analysis of the reported data has been carried out and |

| |the present state-of-art of HPLC for determination of impurities of analgesic, antibiotic, anti-viral, anti-hypertensive, |

| |anti-depressant, gastro-intestinal and anti-neoplastic agents has been discussed.8 |

| | |

| |Ming-Thau Shew stated that a HPLC method using bared silica column eluted with aqueous solvent mobile phase was |

| |developed for determination of tetrahydrozoline hydrochloride in ophthalmic preparations. A mixture of methanol and water |

| |(70:30, v/v) containing 0.03% triethylamine and 0.02% acetic acid was used as mobile phase, and chlorpheniramine maleate |

| |as an internal standard. The flow rate was 1 mL/min and the detection was at 254 nm. The quantitation limit was 1.0 μg/mL.|

| |Average recoveries range from 98.9 to 99.9%. The linearity of the calibration curve of tetrahydrozoline hydrochloride was |

| |well correlated (r2 = 0.9996) within the range from 12.5 to 500 μg/mL as well as from 1.0 to 20 |

| |μg/mL (r2 = 0.9997). This study further reports a simple and quick method for routine quantitative analysis of |

| |tetrahydrozoline hydrochloride in an ophthalmic solution that contains a relatively high concentration of sulfamethoxazole|

| |sodium and methyl paraben.9 |

| | |

| |Suresh Kumar S has stated that simple, precise and economical RP-HPLC method has been developed for the estimation of |

| |Bimatoprost in bulk and in ophthalmic solution. The mobile phase used was a mixture of phosphate buffer (PH 2.8 is |

| |adjusted with orthophosphoric acid) and acetonitrile (55:45 v/v). The wavelength used for detection of Bimatoprost was 210|

| |nm and flow rate 1 ml/min. Linearity was determined for Bimatoprost in the range of 50-250 μg/ml. The correlation |

| |coefficient (‘r’) value was found to be 0.9998. The method was validated with respect to accuracy, precision, linearity |

| |and robustness as per the ICH Guidelines. The proposed method can be successfully used to determine the drug content in |

| |marketed formulation.10 |

| | |

| |Mathrusri Annapurna M has stated that fast, sensitive and accurate reverse phase liquid chromatographic method was |

| |developed and validated for the simultaneous determination of Dorzolamide and Timolol maleate in ophthalmic preparations. |

| |Chromatographic separation was achieved on Inertsil ODS 3V C18 column (250 X 4.6 mm, 5 μm particle size) with mobile phase|

| |consisting of Acetonitrile and 1-Octane Sulphonic acid buffer (0.02M) pH adjusted to 3.5 ± 0.05 with o-phosphoric acid |

| |(36:64 V/V) at a flow rate of 1.0 mL/min. The analytes were detected at 254 nm and 295 nm for Dorzolamide and Timolol |

| |maleate respectively by PDA detector. Brimonidine was used as internal standard (IS). The retention time of Dorzolamide, |

| |Timolol maleate and Brimonidine were found to be at 6.020 ± 0.02, 8.254 ± 0.01 and 4.636 ± 0.01 mins respectively. The |

| |linearity of the method ranged between 4-720 and 1-180 μg/mL for Dorzolamide and Timolol maleate respectively with |

| |correlation coefficient 0.999 for both the drugs in binary mixture. The LOD was found to be 0.6951 μg/mL and 0.2489 μg/mL |

| |for Dorzolamide and Timolol maleate respectively and LOQ was found to be 2.3214 μg/mL and 0.8317 μg/mL for Dorzolamide and|

| |Timolol maleate respectively.11 |

| | |

| |Amit Maru has reported that Dorzolamide Hydrochloride is used to lower the increased intraocular pressure in open-angle |

| |glaucoma and ocular hypertension. Timolol maleate is indicated in the treatment of elevated intraocular pressure in |

| |patients with ocular hypertension or open-angle glaucoma. In this article, a high performance liquid chromatography (HPLC)|

| |method with UV-Visible detector wavelength (254 nm & 295 nm) for Dorzolamide Hydrochloride & Timolol maleate was developed|

| |and validated. The mobile phase consisted of methanol : buffer (60:40), the pH was adjusted to 3 with glacial acetic acid,|

| |was run through a Column C-18, (EC 150/4.6 NUCLEOSIL 100-5), reverse phase analytical column. The experiment was carried |

| |out at room temperature for Dorzolamide Hydrochloride & Timolol maleate. Analytical run time was less than 10 min. The |

| |assay exhibited good linear relationship. Accuracy & Precision were over the concentration range of 10 to 30 mg/ml & 40 to|

| |120 mg/ml for Dorzolamide Hydrochloride and Timolol Maleate respectively. This method was found to be applicable for |

| |determination of the Dorzolamide Hydrochloride & Timolol maleate in active pharmaceutical ingredient (API) and |

| |pharmaceutical product also.12 |

| | |

| |6.3 OBJECTIVE OF STUDY |

| | |

| |The primary objective of the Study is to develop HPLC method and validate it for the estimation of Opthalmic solutions as |

| |shown in the schematics below: |

| |Gather information |

| | |

| |Make a plan |

| | |

| |Optimize retention factor(KI) |

| | |

| |Incomplete resolution Complete resolution |

| | |

| |Change Selectivity |

| | |

| |Incomplete resolution Complete resolution |

| | |

| |Optimize Column efficiency |

| | |

| |Incomplete resolution Complete resolution |

| | |

| |Validate Qualitation |

| | |

| |Fail Pass |

| | |

| |Validate Quantitation |

| | |

| |Fail Pass |

| | |

| |Evaluate and optimize method |

| |For routine use |

| | |

| | |

| |The secondary objective of the study is to validate the developed HPLC method |

| |by evaluating certain validation parameter by ICH Q2B guidelines. |

| |Specificity |

| |Linearity |

| |Precision |

| |Accuracy |

| |Intermediate precision |

| |Limit of detection |

| |Limit of quantitation |

| |Ruggedness |

| |Robustness |

|7 |MATERIALS AND METHODS |

| | |

| |7.1 SOURCE OF DATA |

| | |

| |Data will be collected from literature surveys, abstracts, journal, various related |

| |Website , industry, research publication and from library of Krupanidhi College of |

| |Pharmacy, Indian Institute of Science library Banglore etc. |

| | |

| |METHOD OF COLLECTION OF DATA |

| | |

| |Data will be collected from the following step-wise experimental procedure proposed in study. |

| | |

| |Development of analytical method for estimation of Opthalmic formulation by HPLC. |

| | |

| |By performing experiment in the laboratory from the information obtained from above sources (7.1). |

| |Perform trials with different stationary phase and mobile phase in different ratio with suitable column and sample |

| |concentration and optimization of parameter. |

| |Conclude the various results from above experiments for stating appropriate method to conduct the following study. |

| | |

| | |

| | |

| | |

| | |

| | |

| |Validation of developed HPLC method |

| | |

| |Developing of validation protocol |

| |Defining the application, purpose, and scope of the method |

| |Defining performance parameter and acceptance criteria |

| |Qualify the material |

| |Performing system suitability and/or analytical control checks for the routine |

| |Document validation experiment and result in the validation report. |

| |DOES THE STUDY REQUIRE ANY INVESTIGATIONS OF INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMAN OR ANIMALS? IF SO |

| |PLEASE DESCRIBE BRIEFLY? |

| | |

| |-NO- |

| |HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF AS ABOVE |

| | |

| |Not applicable |

|8 |LIST OF REFERENCES |

| |1. Ahuja S , Scypinski S. Hand book of pharmaceutical analysis. Academic press. 2001;vol3.p.345-86. |

| |2.Vidal-ollivier E, Schwadrohn G, Elias R , Balansard G and Babadjamian A. Determination the ophthalmic drug guaiazulene |

| |by high performance liquid chromatography. J Chrom A. 1989;vol463.p.227-8. |

| |3.Vidal E, Guigues M, Balansard G and Elias R. Determination of ophthalmic therapeutic metipranolol and its degradation |

| |product by reversed-phase high-performance liquid chromatography. J Chrom A. 1985;vol348.p.304-8. |

| |4.Kord A S, Irina T and Matier W L . A novel HPLC method for determination of EDTA in a cataract inhibiting ophthalmic |

| |drugs. J Pharm Biomed Anal.1995;vol13 (4-5).p.575-80. |

| |5. Glickman R D, Davis L T, Kumar N , Nijm L M , Ulanski L J , Elmer Y Tu, Fiscella R G, Peterson R J et.al. An adaptable|

| |HPLC method for the analysis of frequently used antibiotics in Ocular samples. J Chrom B. 2010; vol878.p.2421-6. |

| |6. Carrier A , Labranche L P , Dumont S N , Levesque S . Rapid determination of total benzalkonium chloride content in |

| |ophthalmic formulation. J Pharm Biomed Anal.2007;vol43(1).p.989-93. |

| |7.Gurny.R , Ibrahim.H, Boye.T, Wermeille.M, Buri.P. Determination of pilocarpine by HPLC in presence of isopilocarpine |

| |and a ph-sensitive polymer in ophthalmic dispersions. J Pharm Biomed Anal. 1987; vol5(4).p.379-82. |

| | |

| |8.Rao N R and Nagaraju V. The recent trends in development of HPLC methods for determination of |

| |impurities in drugs. J Pharm Biomed Anal 2003;vol33.p.335-77. |

| | |

| |9.Ming-Thau Sheu , Ming-Chuan Huang , Hsiu-O Ho , Kuo-Ching Wen . HPLC analysis of Tetrahydrozoline hydro chloride in |

| |opthalmics solutions by silica column eluted with aqueous solvent mixtures . J Food Drug Anal 2002;vol10[2].p.88-94. |

| |10. Kumar S S, Natraj K, Asadulla Khan et. al. Development and validation of RP-HPLC method for estimation of |

| |Bimatoprost in pharmaceutical dosage forms . J Pharm Res.2011;vol4[10].p.3733-4. |

| |11. Annapurna M M, Narendra A, Deepika D. Development and validation of RP-HPLC method for simultaneous determination |

| |of Dorzolamide and Timolol maleate in pharmaceutical dosage forms . J Drug Del Ther. 2012;vol2[2].p.81-7. |

| |12. Maru A , Nagori B P, Muysuni P , Gupta S. Method development and its validation for simultaneous estimation of |

| |Timolol maleate and Dirzolamide hydrochloride in as API and in Opthalmic solution dosage forms by RP-HPLC . J Chem Pharm |

| |Res . 2011;vol3[4].p.866-74. |

| |13. Rao R.M, Rao Y.M and Shah A.H. A reverse phase ion-pairing HPLC method for the stability monitoring of sulphacetamide |

| |ophthalmic preparations. J Pharm Biomed Anal.1999; vol20.p. 717-22. |

| |14. Lon Hall and Chadwick V. Quantitative determination of sulfanilamide in sodium sulfacetamide raw material and |

| |ophthalmic solutions by high-performance liquid chromatography. J Chrom A. 1980;vol478.p.438-45. |

| |15. Mehta H P, Fegade J D, Chaudhari R Y , Patil V R . Simultaneous spectrophotometric estimation of Ofloxacin and |

| |Ketorolac Tromethamine in Opthalmic dosage forms . Int J ChemTech Res .2009;vol1.p.189-94. |

| | |

| |16. Ghosh S J , Darbar S, Chakruborty S P. Simultaneous Determination & Validation of Ofloxacin & Ornidazole in combined |

| |dosage pharmaceutical formulation . Int J Pharm Tech Res.2010;vol 2(1).p. 367-374. |

| | |

| |17. Nair R, Badivaddin Md T, Sevukarajan M, Sakeena parveen S, Jeyachitra B. Development and characterization of Ofloxacin|

| |micro emulsions for ocular application . Int J Chem Pharm Sci . 2011;vol2[2]. |

| |18. International Conference on Harmonization, “Q2B: Validation of Analytical procedures; Methodology, Availability.Fed |

| |Regist. 1997; 62(96):27463-76. |

| | |

| 9 |SIGNATURE OF THE CANDIDATE | |

| | | |

| | | |

| | | |

| | | |

| | | |

| | |(Priyanka Seegaragadda) |

|10 |REMARKS OF THE GUIDE |

| | |

| |Developing and validation of an analytical method for a novel formulation is a first step and essential regulatory |

| |requirement in pharmaceutical development because of the novel formualtion’s altered specificity and allied validatory |

| |parameters. This work aims to develop and validate the quantification of a designated active pharmaceutical ingredient of |

| |a novel opthalmic formulation. This work is recommended for necessary approvals. |

| | |

|11 |NAME AND DESIGNATION | |

| |11.1 GUIDE |Chandramouli R |

| | |Assistant Professor & Head |

| | |Department of Quality Assurance |

| | |Krupanidhi College of Pharmacy |

| | |Bangalore 560035 |

| |11.2 SIGNATURE OF GUIDE | |

| | | |

| |11.3 CO GUIDE |-NA- |

| |11.4 SIGNATURE OF CO GUIDE |-NA- |

| |11.5 HEAD OF THE DEPARTMENT |Chandramouli R |

| | |Assistant Professor |

| | |Department of Quality Assurance |

| | |Krupanidhi College of Pharmacy |

| | |Bangalore 560035 |

| |11.6 SIGNATURE OF HOD | |

| | | |

|12 |12.1 REMARKS OF THE PRINCIPAL |

| |The program and research work undertaken by Priyanka Seegaragadda will have potential implication in the field of QUALITY |

| |ASSURANCE. Hence the project is recommended and requested for clearance and approval |

| |12.2 SIGNATURE OF THE PRINCIPAL | |

| | | |

| | | |

| | |DR. PREM KUMAR N |

| | |DEAN |

| | |KRUPANIDHI COLLEGE OF PHARMACY |

| | |BANGALORE 560035 |

| | |KARNATAKA |

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