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“DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIR VALIDATION FOR THE DETERMINATION OF PANTOPRAZOLE SODIUM AND NORFLOXACIN HYDROCHLORIDE IN BULK AND MARKETED FORMULATIONS” MASTER OF PHARMACY DISSERTATION PROTOCOL SUBMITTED TO THE RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE BY DEVADAS SHWETHA M.Pharm-I Under The Guidance of Dr. E.V.S. SUBRAHMANYAM. M.PHARM,Ph.D DEPARTMENT OF QUALITY ASSURANCE, SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143 2012-2014 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA ANNEXURE – II REGISTRATION OF SUBJECT FOR DISSERTATION 1.0 NAME AND ADDRESS OF DEVADAS SHWETHA THE CANDIDATE 1st YEAR M. PHARM, DEPT. OF QUALITY ASSURANCE SRINIVAS COLLEGE OF PHARMACY, VALACHIL,POST PARENGIPETE, MANGALORE -574143 2.0 NAME OF THE INSTITUTE SRINIVAS COLLEGE OF PHARMACY, VALACHIL, PARENGIPETE (POST) MANGALORE-574143 3.0 COURSE OF STUDY & MASTER OF PHARMACY SUBJECT (QUALITY ASSURANCE) 4.0 DATE OF ADMISSION 26TH MAY, 2012 5.0 TITLE OF THE TOPIC: “DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIR VALIDATION FOR THE DETERMINATION OF PANTOPRAZOLE SODIUM AND NORFLOXACIN HYDROCHLORIDE IN BULK AND MARKETED FORMULATIONS” 2 6.0 BRIEF RESUME OF THE INTENDED WORK: 6.1 Need for study: Analytical method development for pharmaceutical formulations: Analytical methods are required to characterize drug substances and drug products composition during all phases of pharmaceutical development. Development of methods to achieve the final goal of ensuring the quality of drug substances and drug products must be implemented in conjunction with an understanding of the chemical behavior and physicochemical properties of the drug substance. These determinations require highly sophisticated instruments and methods like HPLC, HPTLC, Gas Chromatography and Spectrophotometer etc. Extensive literature survey reveals that several analytical methods have been reported for the estimation of Pantoprazole sodium and Norfloxacin hydrochloride in pharmaceutical dosage form which includes Spectrophotometric methods, HPLC and RP-HPLC. Hence there is a need for the development of newer, simple, sensitive, rapid, accurate and reproducible analytical methods for the routine estimation of Pantoprazole sodium and Norfloxacin hydrochloride in bulk and pharmaceutical dosage form. 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. 3 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. The following is a suggested method development scheme for a typical HPLCUV 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 an 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). 4 6.3 DRUG PROFILE OF PANTOPRAZOLE SODIUM:1,2,3 DRUG CATEGORY : Antiulcer, Proton pump inhibitors (PPI’s) CHEMICAL STRUCTURE: PANTOPRAZOLE SODIUM IUPAC NAME: sodium 5- (difluoromethoxy) - 2 – [[(3, 4-dimethoxy-2-pyridinyl) methyl] sulfinyl]-1H-benzimidazole sesquihydrate EMPIRICAL FORMULA: C16H14F2N3NaO4S,11/2 H2O MOLECULAR WEIGHT: 432.4 SOLUBILITY: Freely soluble in water and very slightly soluble in phosphate buffer pH 7.4 and practically insoluble in n-hexane. BIOAVAILABILITY: 77% PROTEIN BINDING: 98% HALF-LIFE: 1 hour EXCRETION: Renal PHYSICAL STATE: White to off white powder. PHARMACOLOGY3 Pantoprazole sodium is a newer H+ K+ ATPase inhibitor. It is more acid stable and has higher bioavailability than omeprazole. It is available for i.v. administration, particularly employed in bleeding peptic ulcer and for prophylaxis of 5 acute stress ulcers. It is used for short-term treatment of erosion and ulceration of the oesophagus caused by gastroesophageal reflux disease. Initial treatment is generally of eight weeks' duration, after which another eight week course of treatment may be considered if necessary. It can be used as a maintenance therapy for long term use after initial response is obtained. Pantoprazole is metabolized in the liver by the cytochrome P450 system. Metabolism mainly consists of demethylation by CYP2C19 followed by sulfation. Another metabolic pathway is oxidation by CYP3A4. Their metabolites are not thought to have any pharmacological significance. It is relatively free of drug interactions; however, it may alter the absorption of other medications that depend on the amount of acid in the stomach, such as ketoconazole or digoxin. Generally inactive at acidic pH of stomach, thus it is usually given with a pro kinetic drug. MECHANISM OF ACTION:4 Pantoprazole is a proton pump inhibitor (PPI) that suppresses the final step in gastric acid production by forming a covalent bond to two sites of the (H+,K+ )ATPase enzyme system at the secretory surface of the gastric parietal cell. This effect is dose related and leads to inhibition of both basal and stimulated gastric acid secretion irrespective of the stimulus. ADVERSE EFFECTS:3 Antacid preparations such as pantoprazole by suppressing acid mediated break down of proteins, leads to an elevated risk of developing food and drug allergies. This happens due to undigested proteins then passing into the gastrointestinal tract where sensitization occurs. Common effects are: Gastrointestinal: abdominal pain (3% ), diarrhoea (4% ), flatulence (4% ) Neurologic: headache (5% ) Serious effects are: Gastrointestinal: atrophic gastritis, clostridium difficile diarrhoea Hematologic: thrombocytopenia (less than 1% ) Immunologic: stevens-johnson syndrome, toxic epidermal necrolysis 6 Musculoskeletal: disorder of muscle, fracture of bone, osteoporosis, hip fracture, rhabdomyolysis Renal: interstitial nephritis, acute (rare). 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. Kumar RS, Pamireddy P, Emmadi CS, Sereya K5 carried out simultaneous determination of naproxen sodium (NAP) and pantoprazole sodium (PAN) in bulk and pharmaceutical dosage form by validated ultra-violet spectrophotometric method. The spectrophotometric method involves the simultaneous equation method at 232.0 nm and 291.0 nm over the concentration range of 10μg/ml for both by using 0.1M NaOH as solvent. The method was validated for linearity, precision, sensitivity, and specificity. The calibration curves were linear over the range of 2-10 μg/ml for both NAP and PAN, with significant high value of correlation coefficient (>0.995 for both drugs). The percentage recovery value for NAP was 100.03% and for PAN was 100.2%. Reddy BPK, Reddy YR, Ramachandran D6 carried out determination of pantoprazole sodium and lansoprazole in individual dosage form tablets by RP HPLC using single mobile phase. The compounds were well separated an isocratically on a C18 column [Use Inertsil C18, 5m , 150 mm x 4.6 mm] utilizing a mobile phase consisting of acetonitrile: phosphate buffer (60:40, v/v, pH 7.0) at a flow rate of 1.0 ml/min with UV detection at 230 nm. The retention time of pantoprazole sodium and lansoprazole was found to be 2.017 min and 2.538 min. The procedure was validated for linearity (Correlation coefficient = 0.999). The study showed that reversed-phase liquid chromatography is sensitive and selective for the determination of pantoprazole sodium and lansoprazole using single mobile phase. 7 Kakde RB, Gedam SN, Chaudhary NK, Barsagade AG, Kale DL, Kasture AV7 carried out three-wavelength spectrophotometric method for simultaneous estimation of pantoprazole(PAN) and domperidone(DOM) in pharmaceutical preparations. The absorbance value at 331nm was used for the estimation of PAN where DOM showed zero absorbance. The absorbance value for DOM was estimated by taking difference of absorbance at two wavelengths 284 nm and 364.5nm. This method obeyed Beer’s law in the concentration range of 10-50 μg/ml for PAN, DOM and their mixture. The results of analysis have been validated statistically and recovery studies confirmed the accuracy of the proposed method. The method was found to be simple, rapid and accurate. Hence can be used for routine simultaneous estimation of these drugs in formulations. Cass QB, Degani ALG, Cassiano NM, Pedrazolli enantiomeric determination of pantoprazole in JJ8 human carried out plasma by multidimensional high-performance liquid chromatography. Multidimensional HPLC is a powerful tool for the analysis of samples of a high degree of complexity. This work reports the use of multidimensional HPLC by coupling a RAM column with a chiral polysaccharide column to the analysis of pantoprazole in human plasma by direct injection. The enantiomers from the plasma samples were separated with high resolution on a tris (3,5dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA octyl column. Water was used as solvent for the first 5 min in a flow-rate of 1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile–water (35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which were detected by UV at 285 nm. Analysis time was 28 min with no time spent on sample preparation. A good linear relationship was obtained in the concentration range of 0.20 to 1.5 μg/ml for each enantiomer. Inter and intraday precision and accuracy were determined by one low (0.24 μg/ml), one medium (0.70 μg/ml) and one high (1.3 μg/ml) plasma concentration and gave a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries of pantoprazole enantiomers were in the range of 93.7–101.2%. 8 Erk N9 studied differential pulse anodic voltammetric determination of pantoprazole in pharmaceutical dosage forms and human plasma using glassy carbon electrode. The best voltammetric response was reached for a glassy carbon electrode in Britton–Robinson buffer solution of pH 5.0 submitted to a scan rate of 20.0 mV s−1 and pulse amplitude of 50.0 mV. This electroanalytical procedure was able to determine pantoprazole in the concentration range 6.0 × 10−6 –8.0 × 10−4 M. Precision and accuracy of the developed method was checked with recovery studies. The limit of detection and limit of quantitation were found to be 4.0 × 10−7 and 9.0 × 10−7 M, respectively. Rapidity, precision, and good selectivity were also found for the determination of pantoprazole in pharmaceutical dosage forms and human plasma. For comparative purposes high-performance liquid chromatography with a diode array and UV/VIS detection at 290.0 nm determination also was developed. Thanikachalam S, Rajappan M, Kannappan V10 studied stability-indicating HPLC method for simultaneous determination of pantoprazole and domperidone from their combination drug product. The proposed HPLC method utilizes Phenomenex® Gemini C18 column (150 mm × 4.6 mm i.d., 5 μm) and mobile phase consisting of methanol-acetonitrile-20mM dipotassium hydrogen phosphate and phosphoric acid buffer pH 7.0 (20:33:47, v/v/v) at a flow rate of 1.19 mL min−1. Quantitation was achieved with UV detection at 285 nm based on peak area with linear calibration curves at concentration ranges 0.5–5.0 μg mL−1 for domperidone and 1.0–10 μg mL−1 for pantoprazole (R 2 > 0.999 for both drugs). The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation and robustness. This method has been successively applied to pharmaceutical formulation and no interference from the tablet excipients was found. Domperidone, pantoprazole and their combination drug product were exposed to acid, base and neutral hydrolysis, oxidation, dry heat and photolytic stress conditions and the stressed samples were analyzed by the proposed method. 9 Raman NVVSS, Reddy KR, Prasad AVSS, Ramkrishna K11 carried out validated chromatographic methods for the determination of process related toxic impurities in pantoprazole sodium. A GC–MS method for the simultaneous determination of two process related toxic impurities viz. 2(chloromethyl)-3, 4-dimethoxypyridine hydrochloride (CDP) and dimethyl sulfate (DMS) and RP-LC for the routine determination of CDP in pantoprazole sodium (PPS) are presented. In GC–MS, a temperature gradient program was performed on a capillary DB-624 column (60 m × 0.32 mm × 1.8 μm). LC analysis of CDP was done on a Novaflex C18 (250 × 4.6 mm, 5 μm) column using mobile phase containing buffer (0.02 M potassium dihydrogen phosphate and 0.0025 M di potassium hydrogen phosphate) and acetonitrile in 46:54 v/v ratio. The flow rate was 1.0 mL min−1 and the elution was monitored at 220 nm. Both methods were validated as per International Conference on Harmonization (ICH) guidelines. GC–MS is able to quantitate up to 3.0 ppm of CDP and DMS whereas with RP-LC up to 9.0 ppm of CDP could be quantitated. Jagatiya V, Patel J12 carried out simultaneous estimation of cinitapride and pantoprazole sodium by Rp-Hplc in their marketed formulation. A reversedphase C-18 column (250 mm × 4.60 mm i.d., particle size 5 μm) with mobile phase consisting of acetonitrile: water: triethylamine (80:20:0.05) was used. The flow rate was 1.2 ml/min and effluents were monitored at 260nm. The retention times of cinitapride and pantoprazole sodium were found to be 5.26 ± 0.10 min and 1.72 ± 0.10 min, respectively. The method was validated in terms of linearity, range, accuracy, and precision, limit of detection (LOD) and limit of quantitation (LOQ). The method showed good linearity in the range of 1228 μgm/ml for cinitapride and 24-56 μgm/ml for pantoprazole sodium. The percentage recoveries of cinitapride and pantoprazole sodium were found to be between 99.52 - 99.85% and 99.38 – 99.74 % respectively. The percentage RSD for the method precision was found to be less than 2%. 10 6.5 DRUG PROFILE OF NORFLOXACIN HYDROCHLORIDE13,14 DRUG CATEGORY: Antibacterial CHEMICAL STRUCTURE: .HCl NORFLOXACIN HYDROCHLORIDE IUPAC NAME: 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3carboxylic acid hydrochloride EMPIRICAL FORMULA: C16H18FN303.HCl MOLECULAR WEIGHT: 355.79 SOLUBILITY: Freely soluble in water and in glacial acetic acid. Bioavailability: 30-40% PROTEIN BINDING: 10-15% HALF-LIFE: 3-4 hours EXCRETION: Renal and Fecal PHYSICAL STATE: A white to pale yellow crystalline powder. PHARMACOLOGY:14 Norfloxacin is a synthetic chemotherapeutic antibacterial agent occasionally used to treat common as well as complicated urinary tract infections and genital tract infections. It is also good for bacterial diarrhoeas. It is not recommended for respiratory and other systemic infections, particularly where gram positive cocci are involved. It is sold under various brand names with the most common being Noroxin and in form of ophthalmic solutions it is known as Chibroxin. It is a first generation synthetic fluoroquinolone (quinolone) developed by Kyorin Seiyaku K.K. (Kyorin). 11 Chibroxin (ophthalmic) is approved for use in children older than one year of age. Norfloxacin was first patented in 1979. Kyorin granted Merck & Company, Inc., an exclusive license, to import and distribute norfloxacin under the brand name Noroxin. The U.S. Food and Drug Administration (FDA) approved Noroxin for distribution in the United States on October 31, 1986. Since the approval of Noroxin in 1986, there have been numerous upgrades to the warning sections of the package inserts, as well as recent restrictions placed upon the use of Noroxin to treat urinary tract infections (UTIs). MECHANISM OF ACTION:15 Norfloxacin is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV, enzymes necessary to separate bacterial DNA, thereby inhibiting cell division. It inhibits the enzyme bacterial DNA gyrase, which nicks double-stranded DNA, introduces negative supercoils and then reseals the nicked ends. This is necessary to prevent excessive positive supercoiling of the strands when they separate to permit replication or transcription. The DNA gyrase consists of two A and two B subunits. The A subunit carries out nicking of DNA, B subunit introduces negative supercoils and then the A subunit reseals the strand. Norfloxacin bind to A subunit with high affinity and interfere with its strand cutting and resealing function. Recent evidence indicates that in gram positive bacteria the major target of norfloxacin action is a similar enzyme topoisomerase IV which nicks and separates daughter DNA strands after DNA replication .Greater affinity for topoisomerase IV may confer higher potency against gram-positive bacteria. The bacteriocidal action probably results from digestion of DNA by exonucleases whose production is signaled by the damaged DNA. In place of DNA gyrase or topisomerase IV, the mammalian cells possess an enzyme topisomerase II (that also removes positive supercoils which has very low affinity for norfloxacin hydrochloride. Hence the low toxicity to host cells. 12 ADVERSE EFFECTS: 16 Diarrhoea Seizure (convulsions) Confusion, hallucinations, tremors, feeling restless, unusual thoughts or behavior, feeling light headed Severe dizziness, fainting, fast or pounding heartbeats Sudden pain, bruising, etc. 6.6 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: Pant M, Dadare K, Khatri NC17 have reported the application of UV spectrophotometric methods for simultaneous estimation of norfloxacin and tinidazole in bulk and tablet dosage forms. Two simple, accurate and reproducible spectrophotometric methods have been developed for the simultaneous estimation of norfloxacin and tinidazole in pharmaceutical dosage forms. The first method involves determination using the vierodt’s method (simultaneous equation method); the sampling wavelengths selected are 273 nm and 319 nm over the concentration ranges of 2.5-20μg/mL and 540 μg/mL for norfloxacin and tinidazole respectively. The second method involves determination using the multicomponent mode method; the sampling wavelengths selected are 273 nm and 319 nm over the concentration ranges of 2.5-20μg/mL and 5-40 μg/mL for norfloxacin and tinidazole respectively. Wankhede SB, Prakash A, Kumari B, Chittange SS18 have reported simultaneous spectrophotometric estimation of norfloxacin and ornidazole in tablet dosage form. Three simple, accurate and economical methods have been 13 developed for the estimation of norfloxacin and ornidazole in tablet dosage form. First method is based on the simultaneous equations, wavelengths selected for analysis were 273.0 nm (λmax of norfloxacin) and 318.5 nm (λmax of ornidazole), respectively, in 0.1N NaOH. Second method is Qanalysis method, based on absorbance ratio at two selected wavelengths 297.0 nm (iso-absorptive point) and 318.5 nm (λmax of ornidazole). Third method is first order derivative spectroscopy using 297.5 nm (zero cross for norfloxacin) and 264.0 nm (zero cross for ornidazole). The linearity was obtained in the concentration range of 4-20 μg/ml and 5-25 μg/ml for norfloxacin and ornidazole, respectively. Maheshwari RK19 carried out application of hydrotropic solubilization phenomenon in spectrophotometric estimation of norfloxacin in tablets. Hydrotropic solubilization technique is one of the techniques used to increase the aqueous solubilities of poorly water soluble drugs. In the present investigation hydrotropic solution of urea (8 M) has been employed as solubilizing agent to solubilize poorly water-soluble drug, norfloxacin from fine powder of its tablet dosage forms for spectrophotometric determination in ultraviolet region. Norfloxacin shows maximum absorbance at 272 nm. Beer’s law was obeyed in the concentration range of 2-14 μg / ml. Results of analysis were validated statistically and by recovery studies. Sebaiy MM, El-Shanawany AA, El-Adl SM, Abdel-Aziz LM and Hashem HA20 carried out rapid Rp-Hplc method for simultaneous estimation of norfloxacin and tinidazole in tablet dosage form. Separation was carried out on a Chromolith® Performance RP-18e (100 x 4.6 mm) using a mobile phase of MeOH: 0.025M KH2PO4 adjusted to pH 3 using ortho-phosphoric acid (20:80, v/v) at ambient temperature. The flow rate was 4 ml/min and maximum absorption was measured at 290 nm. The standard curve was linear in the concentration range of 1-80 μg/mL for both drugs. The retention time of tinidazole and norfloxacin was noted to be 1.2 and 1.6 minutes respectively, indicating shorter analysis time. 14 El-Walily AFM, Belal SF, Bakry RS21 carried out spectrophotometric and spectrofluorimetric estimation of ciprofloxacin and norfloxacin by ternary complex formation with eosin and palladium (II). Both methods are based on the formation of a ternary complex between palladium (II), eosin and the fluoroquinolone in the presence of methyl cellulose, as surfactant. Spectrophotometrically, under the optimum conditions, the ternary complexes showed an absorption maximum at 545 nm, with apparent molar absorptivities of 3.4 × 104 and 2.7 × 104 mol−1 cm−1 and sandell's sensitivities of 1.01 × 10−2 and 1.12 × 10−2 μg cm−2 for ciprofloxacin and norfloxacin, respectively. The solution of the ternary complex obeyed Beer's law in the concentration range 3–10 μg ml−1 for both quinolones. Rege PV, Sathe PA, Salvi VS, Trivedi ST22 carried out simultaneous determination of norfloxacin and tinidazole in combined drug formulation by a simple electroanalytical technique. In present study, a successful attempt has been made to develop a simple method for the simultaneous determination of norfloxacin and tinidazole using linear sweep voltammetry (LSV) technique. Quantification of norfloxacin and tinidazole was done in Britton-Robinson buffer of pH 6.5 using 0.1M KCl as a supporting electrolye. Both norfloxacin and tinidazole exhibit reduction cathodic peak in given pH with peak potential at -1.40V for norfloxacin and -0.48V for tinidazole. 0.1N HCl was used as solvent for the analysis. The parameters used for the method validation are linearity, accuracy, precision, robustness, ruggedness, loss on drying and loss on quantification. El-Brashy AM, El-Sayed MM, El-Sepai FA23 carried out spectrophotometric and atomic absorption spectroscopic determination of some fluoroquinolone antibacterials by ion-pair complex formation with bismuth (III) tetraiodide. These methods have been developed for the determination of levofloxacin (I), norfloxacin (II) and ciprofloxacin (III) in pure form, tablet formulations and spiked human urine. The methods are based on the formation of ion-pair associates between the drugs and the inorganic complex, bismuth (III) 15 tetraiodide. The reaction occurs in acidic medium to form orange-red ion-pair associates which are instantaneously precipitated. The formed precipitates are then filtered off and the residual unreacted metal complex in the filtrate is determined either spectrophotometrically at 453 nm or by AAS at 223.1 nm. The methods permit the determination of the three studied drugs in the range of 5-80 g mL-1. El-Khateeb SZ, Razek SA, Amer MM24 carried out Stability-indicating methods for the spectrophotometric determination of norfloxacin. Two spectrophotometric procedures for the selective determination of norfloxacin (NF) in the presence of its decarboxylated degradant are described. The first depends upon measurement of the pH-induced absorbance difference (delta A) of the drug solution between 0.1 N HCl and 0.1 N NaOH at 280 nm. The second involves chelation of the intact drug with iron (II) in acetate buffer solution (pH 5.7 +/- 0.1) to form a yellow-coloured chelate which absorbs at 358 nm. The two procedures are applied successfully for the determination of the intact drug both in pure form and in tablet form. The two methods retain their accuracy in the presence of up to 62% and 76% degradants, respectively. 6.7 OBJECTIVES OF THE STUDY: In the proposed work, attempt shall be made : To develop a new method for estimation of Pantoprazole sodium. To develop a new method for estimation of Norfloxacin hydrochloride. To apply validated method for the estimation of Pantoprazole sodium and Norfloxacin hydrochloride in pharmaceutical formulation. To develop a validated method according to ICH guidelines. 16 7.0 7.1 MATERIALS AND METHODS: Drug: Pantoprazole sodium and Norfloxacin hydrochloride Reagents: Ferric chloride Folin ciocaltaeau Potassium dichromate Bromothymol blue Indigo carmine 3-methyl-1,2-benzothiazoline hydrazone (MBTH) etc. Method development: All experiments will be carried out in the Department of Quality Assurance. Srinivas college of Pharmacy, Valachil, Mangalore. Pure sample of Pantoprazole sodium and Norfloxacin hydrochloride will be procured from Industries involved in bulk manufacture of this drug. Dosage formulation will be procured from local market. The methods will be developed and validated in Q.A. lab of Srinivas college of Pharmacy. The methods will be first developed, then Validated as per ICH guidelines, then the method will be applied to the formulations. UV spectrophotometer Shimadzu-UV1700 with spectral band width of 2nm and 10nm and matched quartz shall be used for measuring absorbance for Pantoprazole sodium and Norfloxacin hydrochloride solutions. HPLC instrument JASCO ISOCRATIC HPLC-2000 SYSTEM with C18 column shall be used. Colorimeter instruments Systronics Spectrophotometer 166 shall be used. 17 7.2 SOURCES OF DATA: References from library – Srinivas College of Pharmacy, Valachil, Mangalore. www.pharmainfo.net. www.google.com www.sciencedirect.com www.rxlist.com www.pubmed.com www.medline.com www.wikipedia.com 7.3 Does the study require any investigation to be conducted on patients or animals? No 7.4 Has the ethical clearance been obtained from your institution in case of 7.3? Not applicable 18 8.0 LIST OF REFERENCES 1) Indian pharmacopoeia 2010; vol 3:1865 2) http://rxlist.com/protonix-drug.htm 3) en.wikipedia.org/wiki/Pantoprazole 4) www.drugbank.ca/drug/Pantoprazole(DBoo213) 5) Kumar RS, Pamireddy P, Emmadi CS, Koneru S. Simultaneous determination of naproxen sodium and pantoprazole sodium in bulk and pharmaceutical dosage form by validated ultra- violet spectrophotometric method. Int J Chem Anal Sci 2012; 3(10): 1569-72 6) Reddy BPK, Reddy YR, Ramachandran D. Determination of pantoprazole sodium and lansoprazole in individual tablet dosage forms by RP-HPLC using single mobile phase. E-J Chem 2009; 6(2): 489-94 7) Kakde RB, Gedam SN, Chaudhary NK, Barsagade AG, Kale DL, Kasture AV. Three-wavelength spectrophotometric method for simultaneous estimation of pantoprazole and domperidone in pharmaceutical preparations. Int J Pharm Tech Res 2009; 1(2):386-89 8) Cass QB, Degani ALG, Cassiano NM, Pedrazolli JJ. Enantiomeric determination of pantoprazole in human plasma by multidimensional highperformance liquid chromatography. J chromatogr B Analyt Technol Biomed Life Sci 2002; 766(1):153-60 9) Erk N. Differential pulse anodic voltammetric determination of pantoprazole in pharmaceutical dosage forms and human plasma using glassy carbon electrode. Anal Biochem 2003; 323(1):48-53 19 10) Thanikachalam S, Rajappan M, Kannappan V. Stability-indicating HPLC method for simultaneous determination of pantoprazole and domperidone from their combination drug product. Chromatographia 2008; 67(1-2):41-47 11) Raman NVVSS, Reddy KR, Prasad AVSS, Ramkrishna K. Validated chromatographic methods for the determination of process related toxic impurities in pantoprazole sodium. Chromatographia 2008; 68(5-6):481-84 12) Jagatiya V, Patel J. Simultaneous estimation of cinitapride and pantoprazole sodium by Rp-Hplc in their marketed formulation. 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J Pharm Biomed Anal 1998;17(4-5):829-40 21 9.0 SIGNATURE OF THE CANDIDATE (DEVADAS SHWETHA) The candidate is working under my direct supervision 10.0 REMARKS OF THE GUIDE in laboratory of Srinivas College of Pharmacy, Mangalore-574143 DR. E.V.S. SUBRAHMANYAM, 10.1 NAME AND DESIGNATION OF GUIDE PROFESSOR AND HEAD OF DEPARTMENT, DEPARTMENT OF QUALITY ASSURANCE, SRINIVAS COLLEGE OF PHARMACY 10.2 SIGNATURE OF GUIDE DR. E.V.S. SUBRAHMANYAM, PROFESSOR AND HEAD OF DEPARTMENT, 11.0 HEAD OF THE DEPARTMENT DEPARTMENT OF QUALITY ASSURANCE, SRINIVAS COLLEGE OF PHARMACY 11.1 SIGNATURE OF THE HOD 12.0 REMARKS OF THE PRINCIPAL RECOMMENDED AND FORWARDED FOR FAVOURABLE CONSIDERATIONS. 12.1 SIGNATURE OF THE PRINCIPAL DR.RAMAKRISHNA SHABARAYA A. PRINCIPAL AND DIRECTOR, HEAD OF DEPARTMENT, DEPARTMENT OF PHARMACEUTICS SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE. 22 23