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“DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR THE DETERMINATION OF AZITHROMYCIN AND ITS RELATED SUBSTANCES IN OPTHALMIC SOLUTIONS”

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BENGALURU, KARNATAKA

[pic]

BY

P. DEEPAK SAI RAM

DEPARTMENT OF PHARMACEUTICAL ANALYSIS

PES COLLEGE OF PHARMACY

BENGALURU-560050

UNDER THE GUIDANCE OF

Dr. NAGARAJ

PROF. & HEAD

DEPARTMENT OF PHARMACEUTICAL ANALYSIS

PES COLLEGE OF PHARMACY

BENGALURU-560050

(2013-14)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BENGALURU, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

|1. |Name of the candidate and address: |P.DEEPAK SAI RAM |

| | |Department of Pharmaceutical Analysis, |

| | |PES College of Pharmacy, |

| | |50 Feet Road, Hanumanthanagar, |

| | |BSK I-Stage, |

| | |Bengaluru-560050 |

| | | |

| | |Permanent Address |

| | |S/O P.V.Prabhakar |

| | |D.No-11-206, Opp. Agriculture Staff Quarters, |

| | |Perur Post, Tirupati, Chittoor Dist - 517505 |

| | |Andhra Pradesh |

|2. |Name of the institution: |PES College of Pharmacy |

| | |50 Feet Road, Hanumanthanagar, |

| | |BSK I- Stage, |

| | |Bengaluru -560050 |

|3. |Course of study and subject: |MASTER OF PHARMACY IN |

| | |PHARMACEUTICAL ANALYSIS |

| | | |

|4. |Date of the admission: |15/12/2012 |

|5. |Title of the topic: |

| | |

| |“DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR THE DETERMINATION OF AZITHROMYCIN AND ITS RELATED SUBSTANCES IN OPTHALMIC |

| |SOLUTIONS” |

| | |

| | |

| |

|6. BRIEF RESUME OF THE INTENDED WORK: |

| |

|6.1 Need for the study: |

|General Discussion(1,2,3): |

|Blepharitis is a disease consisting of ‘chronic ocular inflammation that involves the eyelid margin primarily and is a common cause of chronic ocular |

|irritation.’ Blepharitis is typically divided into two subsets based on anterior or posterior eyelid involvement. Posterior blepharitis affects the |

|posterior lamella of the eyelid and involves inflammation of the meibomian glands,whereas anterior blepharitis affects the anterior lamella of the eyelid|

|and the eyelashes; either version can be inflammatory or infectious in nature. Each of these conditions can incite or propagate the other; anterior |

|blepharitis, if not treated, can lead to meibomian gland disease, and vice versa.Anterior blepharitis is primarily composed of staphylococcal and |

|seborrheic disease, involving the eyelashes and associated glands. Posterior blepharitis or meibomian gland dysfunction (MGD), involves the posterior lid|

|margin and meibomian system. Patient symptoms may range from mild chronic irritation including itching, burning and foreign body sensation, to more |

|severe discomfort. Chronic blepharitis and associated inflammation of the lid margin can lead to permanent changes including lid margin ulceration, |

|eyelash loss and misdirection, lid margin thickening and scarring and pathologic changes to meibomian gland orifices. |

|Alterations in the lid margin can subsequently deleteriously affect ocular surface health. Secondary ocular surface involvement can manifest as surface |

|inflammation, functional tear deficiency and corneal pathology, ranging from punctuate epithelial erosions or marginal keratitis to peripheral corneal |

|thinning, neovascularization, and rarely perforation. Finally, both anterior and posterior blepharitis can significantly reduce tear quality through |

|degradation of the lipid component, thus further contributing to tear film dysfunction. Staphylococcal blepharitis is thought to be infectious, whereas |

|other forms of blepharitis are more commonly associated with systemic and inflammatory conditions. Azithromycin, a broad-spectrum antibiotic with potent |

|anti-inflammatory activities, has the potential to effectively treat blepharitis.Azithromycin 1% ophthalmic solution, currently the Food and Drug |

|Administration (FDA) approved for the treatment of conjunctivitis caused by susceptible isolates, has been investigated as a possible off-label treatment|

|for chronic blepharitis. This study examines the current evidence for Azithromycin 1% as a treatment for chronic blepharitis and blepharitis-associated |

|dysfunctional tear syndrome(DTS). |

| |

| |

|Specifically, azithromycin has been shown to suppress activation of nuclear factor kappa-light-chain enhancer of activated B cells(NF-kB) and therefore |

|elicit a subsequent decrease in inflammatory cytokine levels such as interleukin-6 (IL-6) and IL-8. Recently published research on the anti-inflammatory |

|properties of azithromycin in ocular tissues also demonstrate decreased corneal inflammation and inflammatory cytokines in a murine model of corneal |

|inflammation treated with topical azithromycin.More specifically, it demonstrated decreased corneal leukocyte infiltration, decreased messenger RNA |

|expression of Interleukin-1b, tissue necrosis factor-a and intercellular adhesion molecule-1 in the cornea of mice with thermal cautery induced corneal |

|inflammation when compared with vehicle alone. This is significant, as tissue necrosis factor-a along with other inflammatory cytokines, have been |

|demonstrated to be increased in DTS, both with and without meibomian dysfunction. |

|Azithromycin: It is an azalide, a subclass of macrolide antibiotics. It is derived from erythromycin, with a methyl-substituted nitrogen atom |

|incorporated into the lactone ring, thus making the lactone ring 15-membered. |

| |

|[pic] |

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|Drug profile: |

|IUPAC NAME: 2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14- |

|heptamethyl-15-oxo-11-{[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-]oxy}-1-oxa-6-azacyclopentadec-13-yl |

|2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranoside. |

| |

|Empirical formula: C37H72N2O12 . |

| |

|Category: Antibacterial. |

| |

|Molecular weight: 748.984 g.mol−1 |

| |

|Solubility: Practically insoluble in water, freely soluble in ethanol and in methylene chloride. |

| |

|Various analytical methods have been reported in the literature for the assay of azithromycin alone or in combination with other drugs in bulk, |

|biological samples and in pharmaceuticals preparations. Procedures involving voltammetry(4), reverse phase high performance liquid |

|chromatography(RP-HPLC)(5), ion pair HPLC with UV detection(6), automatized method with amperometric detection(7), flow-injection spectrophotometric |

|determination using p-chloranil in the presence of hydrogen peroxide(8), spectrophotometric determination using 2,4-dinitrophenylhydrazine(9), ultra |

|performance liquid chromatography–electrospray ionization mass spectrometry(UPLC-MS)(10) and high-performance liquid chromatography (HPLC)-atmospheric |

|pressure chemical ionization mass spectrometry(11) methods have been used for the analysis of azithromycin. There are also few methods like HPLC method |

|for azithromycin quantitation(12), derivative spectroscopic method for simultaneous estimation of azithromycin and its related products(13) and |

|spectrophotometric estimation in tablets(14), comparative potentiometric method using conventional PVC and multi-walled carbon nanotube sensors(15), |

|adsorption technique using stationary mercury and solid electrode(16) and stability-indicating RP-HPLC techniques to determine azithromycin and its |

|related substances.(17) |

|The literature review reveals that no studies for determining azithromycin and its related substances quantitatively in opthalmics was reported, hence |

|this emphasizes the quantitative estimation of azithromycin in opthalmics.In view of the need for a suitable method for routine analysis of azithromycin |

|in opthalmics, an attempt will be made to develop a simple, precise and accurate instrumental analytical method for its estimation. |

|As chromatographic method of analysis is a pre-requisite for the marketing of most of the formulations, Chromatographic methods are being planned to be |

|developed and validate for the choosen drug.Analytical validation is the corner stone of process validation.Without a proven measurement system it is |

|impossible to confirm whether the manufacturing process has done what it purpose to do.Hence, there is a need to validate the new stability indicating |

|analytical method developed. |

| |

|6.2 REVIEW OF THE LITERATURE: |

| |

|Peter VMD et al has reported Current Evidence for Topical Azithromycin 1% Ophthalmic Solution in the Treatment of Blepharitis and Blepharitis- associated|

|Ocular Dryness. |

| |

|Fuad AR, et al has reported analysis of azithromycin and its related compounds by RP-HPLC with UV Detection. |

| |

|Othman AE et al has reported voltammetric determination of azithromycin at the carbon paste electrode. |

| |

|Alaa EG et al has reported optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related |

|compounds. |

| |

|Zi YY et al has reported determination of azithromycin by ion-pair HPLC with UV detection. |

| |

|Miriam EP et al has reported new automatized method with amperometric detection for the determination of azithromycin. |

| |

|Jose LR et al has reported flow-injection spectrophotometric determination of azithromycin in pharmaceutical formulations using p-chloranil in the |

|presence of hydrogen peroxide. |

| |

|Osama HA et al has reported development and validation of a spectrophotometric method for the determination of macrolide antibiotics by using |

|2,4-dinitrophenylhydrazine. |

| |

|Lingyun C et al has reported quantitative determination of azithromycin in human plasma by ultra performance liquid chromatography–electrospray |

|ionization mass spectrometry and its application in a pharmacokinetic study. |

| |

| |

| |

| |

|Fouda HG et al has reported quantitative determination of the antibiotic azithromycin in human serum by high-performance liquid chromatography |

|(HPLC)-atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC-electrochemical method. |

| |

|Karbainov YA et al has reported adsorption of azithromycin dihydrate at stationary mercury and solid electrode. |

| |

|Nawal al-A et al has reported comparative potentiometric study for determination of azithromycin using conventional PVC and multi-walled carbon nanotubes|

|sensors. |

| |

|Jayanna BK et al has reported spectrophotometric estimation of azithromycin in tablets. |

| |

|Nawal AA-A et al has reported comparative potentiometric study for determination of azithromycin using conventional PVC and multi-walled carbon nanotubes|

|sensors. |

| |

|Karbainov Y et al has reported a technique to adsorp azithromycin dihydrate at stationary mercury and solid electrode. |

| |

|Alaa E et al has reported optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related |

|compounds. |

| |

|Luchs J et al has reported the efficacy of topical azithromycin ophthalmic solution 1% in the treatment of posterior blepharitis. |

| |

|6.3 OBJECTIVES OF THE STUDY: |

| |

|To develop new analytical method for the estimation of azithromycin and its related substances in opthalmics. |

|Validate the developed method in accordance with ICH and various other Pharmacopoeial guidelines. |

| |

| |

| |

|7. MATERIALS AND METHODS: |

|7.1 Source of data: |

| |

|Literature survey was done from library of PES College of Pharmacy, |

| |

|The related data was obtained from: |

|Internet facilities at PES College of Pharmacy, Bengaluru. |

|Indian Institute of Science, Bengaluru. |

|Department of Drug testing Laboratories. Bangalore. |

| |

|7.2 Method of collection of data: |

|Analytical method development on estimation of azithromycin in ophthalmic shall be carried out in PES college of Pharmacy, Bengaluru. The analytical |

|methods will be developed by using HPLC, UV visible spectrophotometer. The data so obtained is treated statistically to determine the compliance of the |

|experimental results as per ICH and other Pharmacopoeial guidelines. |

| |

|JOURNALS: |

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|Global Journal of Medical Research. |

|International Journal of Pharmacy and Pharmaceutical Sciences. |

|Journal of Chromatographic science. |

|Journal of Analytical chemical science. |

|International Journal of Research in Pharmaceutical and Biomedical Science. |

|Indian Journal of Pure and Applied Physics. |

|Journal of Pharmacy Research. |

|Journal of Chemical and Pharmaceutical Research. |

|Journal of electrochemical science |

|Journal of Chromatography B: Biomedical Science and Applications |

| |

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| |

| |

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|7.3. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? |

| |

| |

|NOT APPLICABLE |

| |

| |

| |

|7.4. HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION |

|IN CASE OF 7.3? |

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|NOT APPLICABLE |

| |

|8. REFERENCES: |

| |

|Peter V, Kathryn C. Current Evidence for Topical Azithromycin 1% Ophthalmic Solution in the Treatment of Blepharitis and Blepharitis- |

|Associated Ocular Dryness. Int Ophthalmol Clin. 2011;51(4):43-52. |

|Luchs J. Azithromycin in DuraSite for the treatment of blepharitis. Int Ophthalmol Clin. 2010;30(4):681-8. |

|Jodi L. Efficacy of topical azithromycin ophthalmic solution 1% in the treatment of posterior blepharitis.Adv Ther. 2008;25(9):858-70. |

|Othman A, Niveen A. Voltammetric determination of azithromycin at the carbon paste electrode. Indian J. Pure Appl. chem. 2004;62(3):531-8. |

|Fuad A, Maher K. Analysis of Azithromycin and Its Related Compounds by RP-HPLC with UV Detection. J. Chromatogr. Sci. 2010;48(2):86-90. |

|Zi Y, Lin W, Xing T. Determination of azithromycin by ion-pair HPLC with UV detection. J. Pharm. Biomed. Anal.2009;49(3):811-5. |

| |

| |

| |

| |

|Miriam E, Patricia I. New automatized method with amperometric detection for the determination of azithromycin. Indian J. Pure Appl. Chem. |

|2007;72(1):101-5. |

|Jose I, Helena R, Leonardo P. Flow-Injection Spectrophotometric Determination of Azithromycin in Pharmaceutical Formulations Using p-Chloranil in the |

|Presence of Hydrogen Peroxide. Anal. Sci. 2008;24(7):871-6. |

|Osama HA. Development and validation of a spectrophotometric method for the determination of macrolide antibiotics by using |

|2,4-dinitrophenylhydrazine. J AOAC Int. 2007;90(2):364-71. |

|Lingyun C, Feng Q, Yuanyuan M, Famei L. Quantitative determination of azithromycin in human plasma by ultra performance liquid |

|chromatography–electrospray ionization mass spectrometry and its application in a pharmacokinetic study. J. Chromatogr. B: Biomed Sci Appl. |

|2007;855(2):255–61. |

|Fouda H, Schneider R. Quantitative determination of the antibiotic azithromycin in human serum by high-performance liquid chromatography |

|(HPLC)-atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC-electrochemical method. Ther Drug Monit. |

|1995;17(2):179-83. |

|Patricia Z, C Rita C, A Maria A, T Maria T. A new HPLC method for azithromycin quantitation. J. Pharm. Biomed. Anal. 2002;27(5):833–6. |

|Vishal S, Hasumati R. Development and validation of derivative spectroscopic method for simultaneous estimation of cefixime trihydrate and azithromycin |

|dihydrate in combined dosage form. Int. J. Pharm. Bio. Sci. 2012;3(3):14-25. |

|Jayanna B, Nagendrappa G, Gowda N. Spectrophotometric Estimation of Azithromycin in Tablets. Ind. J. Pharm. Sci. 2012;74(4):365–7. |

|Nawal AA-A, Maha FE-T. Comparative Potentiometric Study for Determination of Azithromycin Using Conventional PVC and Multi-Walled Carbon Nanotubes |

|Sensors. Int J Electrochem Sci. 2012;7:8213 - 25. |

|Karbainov Y, Puchkovskaya E, Karbainova S. Adsorption of azithromycin dihydrate at stationary mercury and solid electrode. Bulletin of the Томsк |

|Pоlytеchnic University. 2007;311(3):81-4. |

|Alaa E, Khalid A, Mohammad W, Nasr M, Maisra A. Optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin |

|and its related compounds. J AOAC Int. 2011;94(2). |

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|9. WEBSITE |

|ncbi.nlm. |

|locate/jpba |

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|10. |Signature of the candidate | |

| | |(P. DEEPAK SAI RAM) |

|11. |Remarks of the guide | |

| | |Forwarded for approval |

| | | |

|12. |Name and Designation of | |

| |12.1 Guide |Dr. NAGARAJ |

| | |Professor & Head |

| | |Department of Pharmaceutical Analysis, |

| | |PES College of Pharmacy, |

| | |Hanumanthanagar, |

| | |Bengaluru-560050 |

| | | |

| |12.2 Signature | |

| | | |

| |12.3 Head of the department |Dr. Nagaraj |

| | |Professor & Head |

| | |Department of Pharmaceutical Analysis, |

| | |PES College of Pharmacy, |

| | |Hanumanthanagar, |

| | |Bengaluru-560050 |

| |12.4 Signature | |

| | | |

|13. | | |

| | |Prof. Dr. S. Mohan |

| |Remarks of the Chairman and Principal |Principal & Director, |

| | |PES College of Pharmacy, |

| | |Hanumanthanagar, |

| | |Bengaluru-560050 |

| | | |

| |13.1 Signature | |

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