“DEVELPOMENT AND VALIDATION OF ANALYTICAL METHODS FOR QUANTITATIVE ESTIMATION OF KETOTIFEN AND SALBUTAMOL IN COMBINED DOSAGE FORM”

DISSERTATION PROTOCOL

SUBMITTED TO

RAJIV GHANDI UNIVERSITY OF HEALTH SCIENCE

BANGALORE, KARNATAKA

BY

SATYA VIJAY SINGH

MPHARMA, PART-1

DEPARTMENT OF QUALITY ASSURANCE

NARGUND COLLEGE OF PHARMACY

BANALORE-85

(2011-2013)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / NAME OF THE CANDIDATE
AND ADDRESS (IN BLOCK LETTERS) / SATYA VIJAY SINGH
NARGUND COLLEGE OF PHARMACY, DATTATREYA NAGAR, II MAIN, 100 FEET RING ROAD, BSK III STAGE, BANGALORE-85,
KARNATAKA.
2. /

NAME OF THE INSTITUTION

/ NARGUND COLLEGE OF PHARMACY,
DATTATREYA NAGAR, II MAIN,
100 FEET RING ROAD, BSK III STAGE, BANGLORE-85, KARNATAKA.
3. /

COURSE OF STUDY AND SUBJECT

/ MASTER OF PHARMACY IN
QUALITY ASSURANCE
4. / DATE OF ADMISSION OF COURSE / 1st JULY 2011
5. /

TITLE OF TOPIC

/ DEVELPOMENT AND VALIDATION OF ANALYTICAL METHODS FOR QUANTITATIVE ESTIMATION OF KETOTIFEN AND SALBUTAMOL IN COMBINED DOSAGE FORM
6.
6.1
6.2
6.3
7.
7.1
7.2
7.3
8. / BRIEF RESUME OF THE INDTENDED WORK
NEED FOR THE STUDY
An estimated 300 million people worldwide suffer from asthma, with 250,000 annual deaths attributed to the disease. Workplace conditions, such as exposure to fumes, gases or dust, are responsible for 11% of asthma cases worldwide. About 70% of asthmatics also have allergies [1].
Asthma is a disorder that causes the airways of the lungs to swell and narrow, leading to wheezing, shortness of breath, chest tightness, and coughing.It is thought to caused by a combination of genetic and environmental factor. Treatment of acute symptom is done usually with an inhaled short-acting beta-2 agonist such as Salbutamol. Symptoms canbe prevented by avoiding triggers, such as allergens and irritant, and by inhaling corticosteroids [2].
Ketotifen is a relatively selective, non-competitive histamine antagonist (H1-receptor) and mast cell stabilizer. Ketotifen inhibits the release of mediators from mast cells involved in hypersensitivity reactions. Decreased chemotaxis and activation of eosinophils have also been demonstrated. Ketotifen also inhibits cAMP phosphodiesterase [3].
Salbutamolis a short-acting, selective beta (2)-adrenergic agonist and thus it stimulates beta (2)-adrenergic receptors. Binding of salbutamol to beta (2)-receptors in the lungs results in relaxation of bronchial smooth muscles. It is believed that Salbutamol increases cAMP production by activating adenylate cyclase, and the actions of Salbutamol are mediated by cAMP. Increased intracellular cyclic AMP increases the activity of cAMP-dependent protein kinase A, which inhibits the phosphorylation of myosin and lowers intracellular calcium concentrations. A lowered intracellular calcium concentration leads to a smooth muscle relaxation and bronchodilation. In addition to bronchodilation, Salbutamol inhibits the release of bronchoconstricting agents from mast cells, inhibits microvascular leakage, and enhances mucociliary clearance [4].
Ketotifen is anti histamine, but when used with Salbutamol it will allow these to be taken longer while still being effective. It does so by up regulating the body's beta-adrenergic receptors. It is extremely effective when used with Salbutamol.
Combination of Ketotifen and Salbutamol is used for allergic asthma mainly in childrens. Ketotifen inhibitsthe release of mediators from mast cells involved in hypersensitivity reactions and Salbutamol is a beta(2)-adrenergic agonist and thus it stimulates beta(2)-adrenergic receptors[5].
There is no analytical method reported for simultaneous estimation of Ketotifen and Salbutamol. Hence, investigation of new analytical method is in need for the quantitative estimation of Ketotifen and Salbutamol, in combination in pharmaceutical dosage form.
REVIEW OF LITERATURE
Ketotifen is official in IP 2010 and chemically it is4,9-dihydro-4-(1-methylpiperidin-4-ylidene)-10H-benzo[4,5]cyclohepta[1,2-b]thiophene-10-one hydrogen fumarateand molecular formula of C23H23NO5S Andmolecular weight is 425.5 g/mol[6].It is sparingly soluble in water slightly soluble in methanol, very slightly soluble in acetonitrile[3].

Fig No 1: Chemical Structure of Ketotifen fumarate
Salbutamolis official in IP 2010 and chemically it is(RS)-1-(4-hydroxy-3-hydroxy-methylphenyl)-2-(tert-butylamino)ethanol sulphate and its molecular formula is (C13H21NO3)2H2SO4 and molecular weight is 576.70g g/mol [7]. It is sparingly soluble in water, soluble in ethanol (96%), slight soluble in ether [4].

Fig no 2: Chemical Structure of Salbutamol sulphate
Maithani M, Singh R (2011) developed stability-indicating HPLC method for the simultaneous determination of Salbutamol sulphate and Theophylline in pharmaceutical dosage form using phenomenax C-18 column and mobile phase containing acetonitrile and phosphate buffer in the ratio of 65:32 % v/v (pH4.2±0.02) with flow rate of 1.2ml /min. Elution was monitored at 235nm. The retention time of Salbutamol and Theophylline was found to be 5.33 min and 13.36min and percentage recovery of Salbutamol and Theophylline were found to be 99.41 and 101.11 respectively [8].
Joshi S, Bhatia C, Bal CS, Rawat MSM (2011) worked onsimultaneous analysis of Phenylephrine hydrochloride, Guaiphenesin, Ambroxol hydrochloride and Salbutamol (as Salbutamol sulphate) by use of a validated high-performance liquid chromatographic method using methanol and acetonitrile as a mobile phase in the ratio of 1:1. The flow rate was kept constant at 1.0 ml/min. Detection was done by UV absorbance at 273 nm for Phenylephrine hydrochloride and Guaiphenesin and 225 nm for Ambroxol hydrochloride and Salbutamol. Percentage recovery and RSD were found as, 100.09% and 0.22% for Phenylephrine hydrochloride, 100.43% and 0.50% for Guaiphenesin, 100.91% and 0.70% for Ambroxol hydrochloride, and 100.54% and 0.55% for Salbutamol respectively [9].
Shohan S, Kaushalendra K, Sagar B (2011) developed spectrphotometric and HPLC method for the simultaneous estimation of Salbutamol sulphate and Prednisolone. The spectophotometric method was developed by solving simultaneous equation using 227 nm and 224 nm as λ max for Salbutamol and Prednisolone respectively. Linearity was observed in concentration range of 6-20 µg/ml for Salbutamol as well as for Prednisolone. Second developed method was RP-HPLC method using C18 column (4.6 mm i.d × 250 mm) and acetonitrile: 0.025 Mpotassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) in the ratio of 30:70% v/v as mobile phase. For HPLC method, linearity was observed in the concentration range of 20-100µg/ml for Salbutamol as well as for Prednisolone and drugs were subjected to oxidation, hydrolysis and heat to apply stress condition for degradation studies. Results of analysis were observed to be valided statically and by recovery studies [10].
Patel PA, Dole MN, Shedpure PS, Sawant SD (2011) worked for the development of spectrophotometric estimation of Salbutamol and Ambroxol in bulk and formulation by two methods.First method was based on solving simultaneous equations for the measurement of absorbance at two wavelengths 223 nm and 244 nm the λ max of Salbutamol sulphate and Ambroxol hydrochloride, respectively using 0.1N hydrochloric acid as a solvent. Second method was based on area under curve method, for this method, the wavelength range of 232-217nm was selected for Salbutamol and 252-237nm for Ambroxol. Both the methods were found to be linear in the concentration range of 2-20μg/ml for Salbutamol and 2-40 μg/ml for Ambroxol. Assay values for Salbutamol sulphate and Ambroxol for tablet analysis by both the methods were found in the range of 98.10% to 100.43% [11].
Chitlange SS, Kaushalendra K, Wankhede SB (2011) developed spectrophotometric and HPLC method for the simultaneous estimation of Salbutamol sulphate and Prednisolone in tablet dosage form. Acetonitrile and 0.025M potassium dihydrogen orthophosphate buffer was used as a mobile phase in the ratio of 30:70% v/v with C18 column as a stationary phase. In second method, the concentration of these drugs were found by solving simultaneous equation and the wavelengths selected were 227 nm for Salbutamol and 244 nm for Prednisolone respectively. Linearity was observed in the concentration range of 20-100μg/ml for Salbutamol as well as for Prednisolone by spectophotometric method [12].
Mishra AK, Kumar M, Mishra A, Verma A, Chattopadhyay P (2010) developed UV spectroscopic method for estimation of Salbutamol from tablet formulation. The wavelength maxima for Salbutamol was found to be 276nm in 0.1 N hydrochloric acid and linearity for this method was observed in the range of 10-120µg/ml while, percentage recovery was found to be 98.56±0.238 with the percentage assay of 99.53 [13].
Pai PNS, Rao GK, Murthy MS, Agrawal A, Puranik S (2009) worked on simultaneous determination of Salbutamol sulphate and Bromhexine hydrochloride in tablets by reverse phase liquid chromatographic method using mobile phase containing of acetonitrile, methanol and phosphate buffer in the ratio of 60:20:20% v/v/v with the flow rate of 1ml/min with wakosil-11 C-18 column and UV detection was done at 224nm.The % recovery was found to be in the range of 95-105 for Salbutamol sulphate and 96.2-102.1 for Bromhexine hydrochloride [14].
Shinghvi I, Sachdeva D (2009) developed spectrophotometric methods forthe quantitative estimation of Ketotifen fumarate from its tablet formulation. The developed methods are based on formation of chloroform extractable colored complex of with 2-nitroso- napthol-4- sulphonic acid and rhodizonic acid. The extracted complex of drug with 2-nitroso- napthol-4- sulphonic acid (method-I), showed absorbance maxima at 436.5 nm and with rhodizonic acid (method-II), showed absorbance maxima at 489.5 nm. The linearity range for both the developed methods was observed in the concentration range of 50-250 µg/ml of Ketotifen [15].
Murtaza G, Ahmad M, Madni MA, Asghar MW (2008) developed new reverse phase HPLC method with fluorescent detection for the determination of Salbutamol sulphate in human plasma. RP-C18 analytical column was used as a stationary phase, with flurocent detector operated at excitation 228nm and emission 310nm. Methanol: ammonium phosphate buffer (67mM, pH 3.0): Triethylaminewas used as a mobile phase in the ratio of 50:50:0.02 % v/v/v with the flow rate of 0.7ml/min.The standard curve was observed linear for the range tested (50-80 ng/ml) and coefficient of determination was observed as 0.9989 [16].
Amanlou M, Nazlou MH, Homa A, Effat S, Hassan F (2007) determined Ketotifen fumarate by spectrophotometric method in raw material and pharmaceutical products using Ion-pair formation. The developed method was based on the formation of a colored ionpair complex (11 drug/dye) of Ketotifen and bromocresol green in buffer pH 3 and extraction was done in chloroform. The extracted complex showed absorbance maxima at 423nm. Beer's law was obeyed in the concentration range of 5.15–61.91µg/ml [17].
Ciesielski W, Zakrzewski R, Zlobinska U (2005) developed a colorimetric titration of Ketotifen from tablet formulation by its reaction with iodine in an alkaline medium. In colorimetric titration using biamperometric end-point detection 0.25-2 μmol (77-618 μg) of Ketotifen was successfully determined. The elaborated method was applied to the determination of Ketotifen in drugs [18].
Kazemipour M, Ansari M, Nateghi A (2005) developed and validated a simple HPLC determination of Ketotifen fumarate for quality control purposes. Chromatography was carried out on a C18 (5.0µm, 300mm×4.0 mm) column using acetonitrile: water 45:55 % v/v adjusted to pH 6.5 with acetic acid as mobile phase at a flow rate of 0.8 ml/min and a 299 nm UV detection with an analysis time of 8.5 minutes. The method was linear over the concentration range of 0.5-12µg/ml(r2=0.999). RSD% for both intra and inter day precision were lower than 3%. Recovery of the method was >98% in the mentioned concentration range [19].
Alali FQ, Tashtoush BM, Najib NM (2004) determined Ketotifen in human plasma by LC-MS. Pizotifen was used as an internal standard. An enzyme hydrolysis of conjugated Ketotifen was conducted with a combination of β-glucuronidase and aryl silfatase. After enzyme hydrolysis liquid-liquid extraction was performed as a cleaning step. The quantitative determination was obtained using selected ion monitoring(SIM) LC-MS. Chromatographic condition was a combination of reverse phase gradient system and a switching column technique. The linearity was observed in the 0.5-20 ng/ml with recovery of 98.04% [20].
Parimoo P, Umapathi P, IIang K (2002) developed simultaneous quantitative determination of Salbutamol sulphate and Bromhexine hydrochloric acid in drug preparation by difference spectrophotometry. This method involved the measurements of the absorbance of a solution of the drug mixture in pH 2.0 buffer solution relative to that of an equimolar solution in 0.1 N methanolic NaOH at wavelengths of 310 and 280 nm. The method followed Beer's law in the concentration range of 0–100 μg/ml for Salbutamol sulphate and 0–200 μg/ml for Bromhexine hydrochloric acid at selected wavelengths [21].
Nnane IP, Damani LA and Hutt AJ (1998) developed and validated a stability indicating high performance liquid chromatographic assays for Ketotifen in aqueous and silicon oil formulations using a reversed phase μBondapak C18(30×0.39 cm) column. The mobile phase used was a ratio of phosphate buffer (0.001 M, pH 7.4): methanol: acetonitrile: trimethylamine (29.8:45:25:0.2 % v/v/v/v) at a flow-rate of 1 ml min−1. The eluent was monitored by UV absorption at 299 nm. Silicon oil-based samples were extracted with HCl (0.05 M) using Imipramine as an internal standard. The recovery of Ketotifen and Imipramine was greater than 80%.The assay was shown to be stability indicating by subjecting solutions of Ketotifen in phosphate buffer to heat, oxidative stress and irradiation with ultraviolet light (254 and 369 nm) for upto 8 hr [22].
OBJECTIVES OF THE STUDY:
Review of literature revealed that there is no analytical method reported for the simultaneous estimation of Ketotifen and Salbutamol in pharmaceutical dosage form.
Hence the goals of the present work are
To develop analytical method to estimate Ketotifen and Salbutamol in combined from by UV-Visible spectrophotometer.
To develop and optimize HPLC method to estimate Ketotifen and Salbutamol in combine dosage from.
To validate the developed methods as per ICH guidelines.
MATERIALS AND METHODS:
Materials
Ketotifen, Salbutamol, marketed formulation, double distilled water, chloroform, methanol, potassium dihydrogen orthophosphate buffer,acetonitrile, hydrochloroic acid, orthophosphoric acid, phosphate buffer, chloroform, 2-nitroso- napthol-4- sulphonic acid, rhodizonic acid, ammonium phosphate buffer, triethylamine, bromocresol green buffer, methanolic sodium hydroxide, silicon oil, trimethylamine,etc.
Journals
  • Acta Chromatographica
  • Asian Journal of Pharmaceutics
  • Asian Journal of Pharmaceutical and Clinical Research
  • Indian Journal of Pharmaceutical Sciences
  • International Journal of Pharmaceutical Sciences
  • Journal of Chemical and Pharmaceutical Research
  • Journal of Analytical and Bioanalytical Techniques
  • Journal of Pharmacy Research
  • Research Journal of Pharmacy and Technology
  • Scholars Research Library
Text Books and Pharmacopoeia
  • Beckett AH, Stenlake JB, Practical Pharmaceutical Chemistry, 4th Ed, Delhi, CBS Publisher and Distributors, 1997
  • Sethi PD, Quantitative Analysis of Drugs in Pharmaceutical Formulation, 3rdEd, Delhi: CBS Publisher and Distributors.
  • Higuchi T, Brochman E, Hanseen H, Pharmaceutical Analysis, Delhi: CBS Publisher and Distributors, 2005.
  • Mendham J, Denney RC, Barnes JD, Kthomas MJ, Vogel’s text Book of Quantitative Chemical Analysis, 6thed. Pearson education Pvt Ltd, 2002.
  • The Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare, Published by The Indian Pharmacopoeia commission, Gaziabad,2010.
Internet Browsing
  • .com
MethodofCollectionofData (IncludingSampling Procedures, IfAny)
Procurement of drug samples and marketed formulations.
Solubility determination of Salbutamol and Ketotifen in various solvents and buffers.
Studying the spectra of both the drug in UV-Visible in region different solvents and selecting the solvent for various analytical studies.
Devolpment of analytical method by Simultaneous Equation, Absorption Ratio, Derivative Spectroscopy, Absorption Correction Method, Multicomponent Analysis etc for the estimation of Kitotifen and Salbutamol in combination by UV-Visible spectrophotometer.
Development of HPLC method for estimation of Kitotifen and Salbutamol.
Validation of developed analytical methods as per ICH guidelines.
Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please mention briefly.
- NOT APPLICABLE -
Has ethical clearance been obtained from your institution in case of 7.3?
- NOT APPLICABLE-
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  7. The Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare, Published by The Indian Pharmacopoeia commission, Gaziabad,2010, 3, 2083.
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  9. Joshi S, Bhatia C, Bal CS, Rawat MSM. Simultaneous analysis of Phenylephrine hydrochloride, Guaiphenesin, Ambroxol hydrochloride and Salbutamol sulphate by use of a validated high-performance liquid chromatographic method. Acta Chromatogra.2011;23(1):109-19.[Available from
  10. Sohan S. Kaushalendra K. and Sagar B.Development and validation of Spectrophotometric and HPLC Method for the simultaneous estimation of Salbutamol sulphate and Prednisolone in tablet dosage from. J Anal Bioanal Tech.2011; 2:117. [available from
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  12. Chitlange SS, Kaushalendra K, Wankhede SB. Spectrophotometric and HPLC method for the simultaneous estimation of Salbutamol sulphate and Prednisolone in tablet dosage form. J Anal Bioanal Tech.2011;2:117.[Available from
  13. Mishra AK, Kumar M, Mishra A, Verma A, Chattopadhyay P. Validated UV spectroscopic method for estimation of Salbutamol sulphate from tablet formulation. Arch Apll Sci Res.2010; 2(3):207-11.[ Available from ]
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  20. Alali FQ, Tashtoush BM, Najib NM. Determination of Ketotifen in human plasma by LC-MS. J Anal Bioanal Tech.2004 Jan;34(1):87-94.[Available from
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  22. Nnane IP, Damani LA, Hutt AJ. Development and validation of stability indicating high performance liquid chromatographic assays for Ketotifen in aqueous and silicon oil formulations. Acta Chromatogra. 1998Dec;48:797-802. [Available from

9. / Signature of the candidate / (SATYA VIJAY SINGH)