Rajiv Gandhi University of Health Sciences s121

“METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF LEVAMISOLE IN MARKETED FORMULATIONS BY NEW INSTRUMENTAL METHODS”.

MASTER OF PHARMACY DISSERTATION PROTOCOL, SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.

BY MR.JOHNSON MISQUITH M.PHARM – I

Under The Guidance Of MRS. PADMAVATHI P. PRABHU

DEPARTMENT OF QUALITY ASSURANCE.

SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143. 2010 – 2012 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE JOHNSON MISQUITH AND ADDRESS: S/O IVAN MISQUITH PLANTERS LANE, KADRI ROAD, KODIALBAIL POST, MANGALORE-575003.

2. NAME OF THE INSTITUTE: SRINIVAS COLLEGE OF PHARMACY, VALACHIL, FARANGIPETE (POST), MANGALORE-574143.

3. COURSE OF THE STUDY AND MASTER OF PHARMACY SUBJECT: (QUALITY ASSURANCE)

4. DATE OF ADMISSION TO THE 31st May 2010 COURSE:

5. TITLE OF THE TOPIC:

“METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF LEVAMISOLE IN MARKETED FORMULATIONS BY NEW INSTRUMENTAL METHODS”. 6. 6.1 Need for the Study : Several spectrophtometric methods have been developed for quantitative determination of levamisole in pharmaceutical formulations on sheep muscle tissue and Photo diode array detection. Levamisole is novel in market and not much data is available on analytical methods by UV and HPLC method. Our intension is to develop economic, less time consuming method for the estimation of levamisole by different analytical methods such as UV and HPLC.

6.2 DRUG PROFILE: Levamisole Chemical Structure:

N H S N

IUPAC Name: (S)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,1-b][1,3]thiazole.

Empirical formula: C11H12N2S, Physical state: White to off-white crystalline powder (light yellow crystalline powder)1, Molecular mass : 204.292 g/mol, Density: 1.31 g/ml, M.P.: 230°C (446 °F), pH: 3.0 – 4.5 (5% aqueous Solution), Solubility: Soluble in water, methanol, slightly soluble in ethanol, very slightly soluble in chloroform, insoluble in acetone1, Stability: Stable under ordinary conditions. Light sensitive.

6.3 Objective of the Study:  To determine λ max in different solvent and pH condition for Levamisole.  To develop simple, selective, rapid, precise and economical analytical method for the estimation of levamisole.  To validate method in terms of accuracy, precision, linearity, limit of detection, limit of quantitation etc.

Levamisole is an anthelminthic and immunomodulator belonging to a class of synthetic imidazothiazole derivatives, used in the treatment of many nematodes particularly in veterinary applications which are effective against Roundworm and Hookworm2. It is available in single pack tablet form of 50mg and 150mg strength (Brand Name: Dewormis, Dicaris). Levamisole is not official in any Pharmacopoeia. An extensive literature survey revealed that only Determination of Levamisole in Sheep Muscle Tissue by High- Performance Liquid Chromatography and Photo Diode Array Detection was carried out. Therefore it was decided to develop a HPLC method for estimation of Levamisole in its marketed formulation.

Pharmacopoeias are replacing the chemical methods and Spectrophotometric methods by more sensitive and accurate HPLC methods for the analysis of drug as well as their formulations. Hence it was thought proper to develop a validated HPLC method for analysis of Levamisole in its marketed formulation.

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.  Tyrpenou AE, Xylouri-Frangiadaki EM3 have developed a high-performance liquid chromatographic method for the determination of levamisole (LVM) residues in sheep

muscle tissue is described. Chromatography was performed on a Zorbax®SB-C18 column

at 50oC and detection by a Photo Diode Array detector monitored at λ max 220 nm. The mobile phase was a mixture of 0.1 % trifluoroacetic acid (v/v) pH 2.0 and acetonitrile- methanol 3 : 2 (v/v) in a combination of 30 : 70 (v/v) and a flow rate of 1.0 mL min-1, delivered isocratically.  Marc C, Siegrid DB, Siska C, Patrick DB4 have developed and validated an LC-MS-MS method with atmospheric pressure chemical ionization for the quantitative determination of levamisole. A liquid-liquid back extraction procedure using hexane-isoamylalcohol (95:5, v/v) as extraction solvent was followed by a solid-phase extraction procedure using an SCX column to clean up the tissue samples. Methyl levamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher 60 RP- select B (5 μm) column using a mixture of 0.1 M ammonium acetate in water and acetonitrile as the mobile phase.  Milada D, Magda T5 developed a LC of the enantiomeric purity of levamisole using stationary phase with bonded naphthylethylcarbamoylated-β-cyclodextrin. The elaborated method used S-naphthylethylcarbamoylated -β-cyclodextrin stationary phase in reversed-phase mode. The optimized mobile phase composition was acetonitrile-0.5% triethylammonium acetate buffer, pH 5.0 (2:8, v/v).  Fouad C, Abdel I, Salvatore C, Audrey T, Joel S, Robert R,6 studied the stability of levamisole oral solutions prepared from tablets and powder. Levamisole concentrations were determined in duplicate by a stability-indicating HPLC method at 0, 1, 2, 3, 4, 7, 14, 30, 60 and 90 days. The initial and final pHs of solutions were measured. And it was conclude that levamisole oral solutions can be refrigerated for 90 days.  James FC, Rhoda A, Donna A, Chris F, Barry S, Pauline W,7 found that the addition of levamisole resulted in more toxicity than 5-FU and leucovorin alone but the addition of levamisole resulted in more immunomodulation than 5-FU and leucovorin alone as evidenced by release of neopterin from monocytes.The maximum tolerated dose of levamisole was (354 mg/m2 ).  Martin PJ, Le Jambre LF8 in 1979 developed a method for detecting levamisole and morantel tartrate resistance by determining the percentage of paralysed third stage larvae in serial dilution of anthelmintic.  Olivia B, Janelle KM, Laure G, Michael JGS 9 in 2008 inferred short and long-term efficacy of levamisole as adjunctive therapy in childhood nephrotic syndrome.  Golub SH, Holmes EC10 in 1979 studied the in vitro assay of immunocompetence in patients with lung cancer treated with levamisole, the results showed that lung cancer patients were depressed in assays of lymphocyte proliferation induced by either nitrogens or allogeneic cells. 7. 7.1 Materials and Methods: Drug: Levamisole. Solvents: acetonitrile, acetone, ethanol, methanol, HCl, Chloroform. Instrument details: Shimadzu, UV-1700, Jasco Isocratic HPLC-2000 System, HPLC Pump PU – 2080, UV/Vis Detector – 2075, Rheodyne Injector 7725, HPLC Column C18. The standard drug will be requested from the industries in India depending on the availability of facilities, one of the following method will be applied for the estimation of levamisole.

1) UV Method: Standard stock solution can be prepared by dissolving 100mg of levamisole in 100 ml of water and then final volume of solution made upto 100 µg/ml of levamisole in a volumetric flask. Solutions containing 20 µg/ml of levamisole can be prepared and scanned in UV region for λ max. For the compound levamisole λ max can be obtained, By dilution of Standard drug in 5,10,15,20 ….50 µg/ml of levamisole and scanned at 200-400 nm. The values of absorbances can be recorded. From this data absorptivity and molar absorbance can be determined for levamisole. By doing this we can estimate the purity of drug in formulation by preparing known concentrations of drug and determining by UV method. 2) HPLC Method: Mixed solutions consisting of 100 µg/ml of levamisole can be prepared in different combinations of solvents using acetonitrile, water, acetone, methanol. Then, eluting the compound solution from column and recording the absorbance parameter by detector. By observing AUC, Peak area we can estimate the amount of drug present in sample. All the validation parameters such as LOD, LOG, Linearity, Precision and Accuracy can be validated using ICH Q2b guidelines. The above method likely to change depending on the observant recorded time to time. 7.2 Source of Data: Data will be obtained from the publication of USFDA, WHO, PICS, MHRA guidelines as well as from Srinivas College of Pharmacy library, Helinet, Science Direct, Ovid, Springer, Blackwell and other internet facility. 7.3 Does the study require any investigations or interventions to be conducted On patients or other human or animals? If so please describe briefly: -- Not applicable--

7.4 Has the Ethical Clearance been obtained from your Institution in case of 7.3? -- Not applicable--

8. LIST OF REFERENCES:

1) Available From: URL:http://www.bikudo.com/product_search/details/280640/levamisole_hydrochlori de.html 30/11/2010 22.23 2) Tripathi KD. Essentials of Medical Pharmacology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2008. 3) Tyrpenou AE, Xylouri-Frangiadaki EM. Determination of levamisole in sheep muscle tissue by high-performance liquid chromatography and photo diode array detection. Chromatographia 2006; 63:321–32. 4) Marc C, Siegrid DB, Siska C, Patrick DB. Quantitative analysis of levamisole in porcine tissues by high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry. J Chromatogra B 2000;742(2):283-93. 5) Milada D, Magda T. LC of the enantiomeric purity of levamisole using stationary phase with bonded naphthylethylcarbamoylated-β-cyclodextrin. J Pharm Biomed Anal 2001; 25:407–15.

6) Fouad C, Abdel I, Salvatore C, Audrey T, Joel S, Robert R, et al. Stability of

levamisole oral solutions prepared from tablets and powder. J Pharm Pharm Sci 2005; 8(2):322-5.

7) James FC, Rhoda A, Donna A, Chris F, Barry S, Pauline W, et al. A phase i study of 5-fluorouracil, leucovorin and levamisole. Cancer Chemother Pharmacol 1997; 39:300-6. 8) Martin PJ, Le Jambre LF. Larval paralysis as an in vitro assay of levamisole and morantel tartrate resistance in ostertagia. Vet Sci Commun 1979; 3(2):159-64. 9) Olivia B, Janelle KM, Laure G, Michael JGS. Short and long-term efficacy of levamisole as adjunctive therapy in childhood nephrotic syndrome. Pediatr Nephrol 2008; 23:575–80. 10) Golub SH, Holmes EC. In vitro assay of immunocompetence in patients with lung cancer treated with levamisole. Cancer Immunol Immunother 1979; 7:143-9. 9. Signature of the Candidate:

(JOHNSON MISQUITH) ((( 10. Remarks of the Guide: The candidate is working under my direct supervision in laboratories of Srinivas College of Pharmacy, Mangalore-574143. 11. 11.1 Name & Designation of the Guide :

MRS PADMAVATHI P. PRABHU M.Pharma (Ph.D) ASSISTANT PROFESSOR, DEPARTMENT OF QUALITY ASSURANCE, SRINIVAS COLLEGE OF PHARMACY.

11.2 Signature of Guide:

(MRS PADMAVATHI P. PRABHU)HINI R. M.) 11.3 Co-Guide:

11.4 Signature of Co-Guide:

11.5 Head of the Department: PROF. Dr. E.V.S SUBRAHMANYAM DEPARTMENT OF QUALITY ASSURANCE, SRINIVAS COLLEGE OF PHARMACY. 11.6 Signature of HOD:

(Dr. E.V.S SUBRAHMANYAM)

12. 12.1 Remark of the Principal:

FORWARDED AND RECOMMENDED FOR FAVORABLE CONSIDERATION.

12.2 Signature of the Principal:

(Dr. A.R SHABARAYA)