RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE –ІІ
REGISTRATION OF SUBJECT FOR DISSERTATION
1.0 / NAME OF THE CANDIDATEADDRESS / PATEL NIRAJKUMAR MUKESHKUMAR
AT & POST- PARAVAT,
TALUKA- HANSOT,
DISTRICT- BHARUCH.
GUJARAT- 394810.
2.0 / NAME OF THE INSTITUTION / AL-AMEEN COLLEGE OF PHARMACY,
BANGALORE.
3.0 / COURSE OF STUDY
SUBJECT / MASTER OF PHARMACY (PART-I)
DEPARTMENT OF QUALITY ASSURANCE
4.0 / DATE OF ADMISSION / 1ST JUNE 2010
5.0 / TITLE OF THE TOPIC:
DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AMOXICILLIN TRIHYDRATE AND CARBOCISTEINE IN CAPSULES
6.0.
6.1.
6.2.
6.3. / BRIEF RESUME OF THE INTENDED WORK
NEED FOR THE STUDY:
· Pharmaceutical products formulated with more than one drug, typically referred to as combination products in single dosage forms are intended to meet previously unmet patient`s need by combining the therapeutic effects of two or more drugs in one product. These combination products can present challenges to the analytical chemist and simultaneous estimation of individual components in combined dosage form plays an important role in the field of pharmacy1.
· One such combination product introduced into the market recently is of Amoxicillin trihydrate and Carbocisteine which is used in respiratory tract infections associated with excessive and/or viscous mucus.
· From the literature survey, it was found that many methods have been reported for estimation of Amoxicillin trihydrate and Carbocisteine individually and in combination with other drugs. Hence no HPLC method for simultaneous estimation of Amoxicillin trihydrate and Carbocisteine has been reported so far.
· Hence there is a need to develop a new HPLC method for simultaneous estimation of Amoxicillin trihydrate and Carbocisteine in capsules.
INTRODUCTION:
· Single dosage forms with combination of drugs are widely used today due to their advantages and their simultaneous estimation of individual component is a challenging task.
· The combination of Amoxicillin trihydrate and Carbocisteine is emerging as one of the widely prescribed combination in single dosage form for the management of respiratory tract infections associated with excessive and/or viscous mucus. A few new brands of this combination are available in the market are CARBOMAX (WINMEDICARE), CIDORESP (SANOFI-AVENTIS), MOXYCARB (PIRAMAL HEALTHCARE) and MUCOBRON (ELDER) 2.
· AMOXICILLIN TRIHYDRATE3,4,5, a β-lactum antibiotic, chemically is (2S, 5R, 6R)[[(2R)-2-amino-2(4 hydoxyphenyl) acetyl] amino]-3, 3-dimethyl-&-oxo-4-thia-1azabicyclo [3.2.0] heptanes-2-carboyic acid with molecular formula of C16H19N3O5S, 3H20 and molecular weight of 419.4. It is highly active against broad spectrum of bacteria used for the treatment of respiratory tract infections.
· CARBOCISTEINE6, a mucolytic agent, chemically is (R)-2-Amino-3-(carboxymethylsulfanyl) propanoic acid with molecular formula C5H9NO4S and molecular weight of 179.2 used in respiratory disorders associated with productive cough.
· In our proposed work, an attempt shall be made to develop and validate a HPLC method for simultaneous estimation of Amoxicillin trihydrate and Carbocisteine in formulation (capsules).
OBJECTIVES OF THE STUDY:
In the proposed work, attempt shall be made :
· To develop a HPLC method for simultaneous determination of Amoxicillin trihydrate and Carbocisteine.
· To validate the developed HPLC method using various analytical parameters such as accuracy, precision, specificity, linearity, range, limit of detection, limit of quantification robustness and ruggedness.
· To apply the validated method for estimation of Amoxicillin trihydrate and Carbocisteine in marketed formulation (capsules).
7.0. / REVIEW OF LITERATURES:
An extensive survey was carried out for the estimation of Amoxicillin trihydrate and Carbocisteine in bulk and marketed dosage forms. It was found that a few methods have been reported for these drugs individually or in combination with other drugs which are presented below.
· Abdulqawi AN et al7 (2010) have reported a reversed-phase HPLC Method for the determination of Amoxicillin Trihydrate in human plasma and Cefadroxil was used as the internal standard. The analytical method was validated and the linearity was within the range 1-80 µg/ml. The LOD and LLOQ for amoxicillin in plasma were 0.080µg/ml and 0.16µg/ml respectively. Intraday accuracy of amoxicillin in plasma ranged from 103.00 ± 8.70 % to 109.82 ± 1.39 % while inter-day accuracy was between 102.00 ± 0.98 % and 110.30 ± 0.36 %. Amoxicillin and Cefedroxil were found to be stable in human plasma samples for short term stability at room temperature and long term stability at -20⁰c. The developed method of analysis provided a sensitive and specific assay for amoxicillin in human plasma.
· Rele RV and Patil SP8 (2010) have reported a reversed Phase High Pressure Liquid Chromatography technique for determination of Carbocisteine from pharmaceutical formulation. The separation of drug was achieved on (Amino
Propyl Silane) APS-2 hypersil (250 X 4.6 mm) 5μ column. The mobile phase consists of a mixture of buffer 0.02 M potassium dihydrogen phosphate monobasic and methanol (65:35 v/v) and detected at 210 nm. The method has been successfully used to analyze Carbocisteine from pharmaceutical formulation.
· Gaurav Tiwari et al9 (2009) have reported two methods for simultaneous estimation of Metronidazole and Amoxicillin in synthetic mixture by Ultraviolet Spectroscopy. First method employs formation and solving of simultaneous equation using 319 nm and 273.8 nm as two analytical wavelengths for both drugs in phosphate buffer pH 7.4. The second method is Q value analysis based on measurement of absorptivity at 319 nm and 289 nm (as an iso-absorptive point). Metronidazole and Amoxicillin at their respective λmax 319 nm and 272 nm and at isoabsorptive point 289 nm shows linearity in a concentration range of 10-50 mcg/mL.The results of analysis have been validated statistically.
· Ch BVN Raju et al10 (2009) have reported that RP-HPLC Method for analysis of related Substances in Amoxicillin drug substance forced degradation studies were conducted on Amoxicillin drug substance using ICH stress study guidelines to demonstrate the specificity and stability-indicating nature of the method. The method was fully validated in accordance with ICH analytical method validation guidelines. The results of the study prove the method is specific, precise, linear, robust, and can be used for evaluation of the stability of amoxicillin drug substance.
· Chandrakant GB et al11 (2009) have reported the development and validation of RP-HPLC method for simultaneous estimation of Amoxicillin trihydrate and Flucloxacillin sodium in capsule dosage form. The separation was made by a Kromasil C18 column (250 cm × 4.6 mm, 5μm) using 0.020 M potassium dihydrogen orthophosphate - acetonitrile (75:25) as mobile phase. The limits of quantification were approximately 0.16μg/ml for amoxicillin trihydrate and 0.25μg/ml for flucloxacillin sodium. Due to its simplicity and accuracy, the method was suitable for routine pharmaceutical quality control.
· Prakash K et al12 (2008) have reported Spectrophotometric estimation of Amoxicillin trihydrate in bulk and pharmaceutical dosage form using citro phosphate buffer pH 7.2. Molar absorptivity, Correlation coefficient and Sandell’s sensitivity values were found to be 1.0020 x 104 mol-1 cm,-1 0.9996 and 0.03906 μg cm-2 respectively. The proposed method has been successfully applied for analysis of the bulk drug and its tablet dosage form. The method has been statistically evaluated and was found to be precise and accurate.
· Naser T et al13 (2006) have reported the development and validation of a simple HPLC method for simultaneous in vitro determination of Amoxicillin and metronidazole at single wavelength (254 nm) in order to assess drug release profiles and drug–excipients compatibility studies for a new floating-sustained release tablet formulation and its subsequent stability studies.
· Michal D et al14 (2004) have reported rapid determination of Amoxicillin in premixes by HPLC. The ground premix samples were extracted for 10 min using 100 ml extraction mixture water–methanol (800:200, v/v). The method was validated for specificity, linearity, solution stability, accuracy, precision, limit of detection, and limit of determination. The detector response for Amoxicillin was linear over the selected concentration range from 2.0 to 40.0 mg ml−1 with a correlation coefficient 0.9999. The mean accuracy was 100.1% with a standard deviation of 0.6%. The limit of detection and the limit of determination were 0.1 and 0.3 mg ml−1, respectively.
· Walash MI et al15 (2004) have reported a high sensitive Fluorimetric determination of Carbocisteine and Ethionamide in drug formulation .The method was based on the reaction of Carbocisteine and Ethionamide with Roth’s reagent (o-phthaldehyde) to get a highly fluorescent isoindole product which emits strong fluorescence at 431 nm and 424 nm after excitation at 329 nm and 339 nm for Carbocisteine and Ethionamide, respectively. The different experimental parameters affecting the intensity of the fluorescence were carefully studied and incorporated into the procedure. The method was successfully applied for determination of Carbocisteine and Ethionamide in their dosage forms.
· Walash MI et al16 (2004) have reported Spectrophotometric determination of Penicillamine and Carbocisteine based on formation of metal complexes with Ni, Co and Pb ions in acetate buffer pH of 6.3, 6.5 and 5.3, respectively, and Carbocisteine with Cu and Ni ions in borate buffer pH of 6.7; 1–70 μg/ml of these drugs could be determined by measuring the absorbance of each complex at its specific λmax. The results obtained are in good agreement with those obtained using the official methods. The method was successful applied for the determination of these compounds in their dosage forms.
· Mei-chich hsu and Pei-wen hsu17 (1992) have reported a reverse phase High-Performance Liquid Chromatographic Method for potency determination of Amoxicillin in Commercial Preparations and for Stability studies. The high-performance liquid chromatographic assay results were compared with those obtained from a microbiological assay of bulk drug substance, capsule, injection, and granule formulations containing Amoxicillin and degraded Amoxicillin. At the 99% confidence level, no significant inter-method differences were noted for the paired results. The results indicated that the proposed method is a suitable substitute for the microbiological method for assays and stability studies of Amoxicillin preparations.
7.1. / MATERIALS AND METHODS:
SOURCES OF DATA:
· References from library - Al-Ameen College of Pharmacy, Bangalore.· www.pharmainfo.net.
· www.jkscience.org
· www.sciencedirect.com
· www.encyclopedia.com
· www.findarticles.com
INSTRUMENTATION:
· UV spectrophotometer Shimadzu - UV1700 with spectral band width of 2mm and 10mm matched quartz will be used for making absorbance readings of Amoxicillin Trihydrate and Carbocisteine.
· The HPLC Shimadzu – LC 10AT, SPD10A detector for isolation and detection of Amoxicillin Trihydrate and Carbocisteine.
· Single pan analytical balance ACCULAB (ALC – 210.3) for weighing.
7.2. / METHODOLOGY:
v The proposed project work shall be carried out in the Quality Assurance departmental laboratory at Al-Ameen College of pharmacy, Bangalore.
v Pure sample of Amoxicillin Trihydrate and Carbocisteine shall be procured from industries involved in the manufacture of these drugs.
v Formulations (Capsules) for the project work shall be procured from the local market.
v A HPLC method shall be developed and validated for simultaneous estimation of Amoxicillin trihydrate and Carbocisteine as per the method development scheme.
· 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.
· To develop preliminary HPLC conditions to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments.
· Develop a suitable sample preparation scheme for the drug product
· Determine the appropriate standardization method and the use of relative response factors in calculations.
· Identify the “weaknesses” of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples.
· Complete method validation with various parameters like Specificity, Linearity, Range, Accuracy, Precision, Detection limit, Quantitation limit, Robustness and System suitability testing according to ICH guidelines 18.
v The developed and validated method shall be applied for determination of Amoxicillin trihydrate and Carbocisteine in formulation (capsules).
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-
8.0. / REFERENCES:
1. Rashmin. An Introduction to Analytical Method Development for Pharmaceutical Formulations. Pharmaceutical Reviews, 2008; 6 (4).
2. CIMS, CMPMedica India private limited, India. April-June 2010; 109:300.
3. Martindale: The complete drug reference, the pharmaceutical press, UK. 2009; 36thed, 1:202-203.
4. British Pharmacopoeia, British pharmacopoeia commission, London, UK. 2001; 1: 305.
5. Hassempour A, Rafati H, Adlnasab L, Bashour Y, Ebrahimzadeh H, Erfan M. Investigation of the Solid State Properties of Amoxicillin Trihydrate and the Effect of Powder pH, AAPS PharmSciTech. 2007;8(4).
6. Martindale: The complete drug reference, the pharmaceutical press, UK. 2009; 36thed, 1:1553-54.
7. Abdulqawi AN, Majed N, Hussein A, Abdussalam IAT, Abdulhafeed A, Naser Z, Abdulmoniem A. Validation and Application of a Reversed-phase HPLC Method for the determination of Amoxicillin Trihydrate in Human Plasma. J.Appl.Sci.Res.2009; 5(12): 2219-24.
8. Rele RV, Patil SP. Reversed Phase High Pressure Liquid Chromatography Technique for Determination of Carbocisteine from Pharmaceutical Formulation. J.Chem.Pharm.Res.2010; 2(4):24-30.
9. Gaurav T, Ruchi T, Brijendra S, Awani KR, Kamla P. Simultaneous Estimation of Metronidazole and Amoxicillin in Synthetic Mixture by Ultraviolet Spectroscopy. Asian J. Research Chem.2008; 1(2): 91-94.
10. Raju Ch BVN, Sharma HK, Rao ChS, Rao GN. RP-HPLC Method for Analysis of Related Substances in Amoxicillin Drug Substance. Acta Chromatographica.2009; 21(1):57–70.
11. Chandrakant GB, Dhiraj SN, Surana SJ, Venkateshwarlu G, Dekate PG. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Amoxicillin trihydrate and Flucloxacillin sodium in capsule dosage form. Int. J. PharmTech Res.2009; 1 (3):935-39.
12. Prakash K, Raju PN, Shantakumari K, Lakshminarasu M. Spectrophotometric Estimation of Amoxicillin Trihydrate in Bulk and Pharmaceutical Dosage Form. E-Journal of Chemistry.2008; 5(S2):1114-16.
13. Naser T, Jaleh V, Farid D, Mohammad RZ. Development and validation of a simple HPLC method for simultaneous in vitro determination of Amoxicillin and Metronidazole at single wavelength. J.pharm Biomed Anal.2007; 43:325–29.
14. Michal D, Romana H. Rapid determination of amoxicillin in premixes by HPLC. J.pharm Biomed Anal.2005; 37:373–377.
15. Walash MI, Amina M ElB, Mohamed ESM, Amina AA. Fluorimetric determination of Carbocisteine and ethionamide in drug formulation. Acta Chim.Slov.2004; 51:283−91.
16. Walash MI, Amina M ElB, Mohamed ESM, Amina AA. Spectrophotometric determination of penicillamine and Carbocisteine based on formation of metal complexes. IL FARMACO.2004; 59:493–503.
17. Mei-chich hsu, Pei-wen hsu. High-Performance Liquid Chromatographic Method for Potency determination of Amoxicillin in Commercial Preparations and for Stability Studies. Antimicrobial agents and Chemotherapy.1992; 1276-79.
18. http://www.ich.org/LOB/media/MEDIA417.pdf [cited 2010 October 12].
9.0 / SIGNATURE OF THE CANDIDATE
10.0 / REMARKS OF THE GUIDE / Forwarded for Approval
11.0 / NAME AND DESIGNATION OF GUIDE / Dr. Mubeen G
Professor
Dept of Quality Assurance
Al-Ameen college of pharmacy, Hosur road, Bangalore – 560027.
11.1 / SIGNATURE
11.2 / CO-GUIDE / -
11.3 / SIGNATURE / -
11.4 / HEAD OF THE DEPARTMENT / Dr. Sanjay Pai P N
Professor and Head,
Dept of Quality Assurance
Al-Ameen college of pharmacy, Hosur road, Bangalore – 560027.
11.5 / SIGNATURE
12.0 / REMARKS OF THE PRINCIPAL / Forwarded for approval
12.1 / SIGNATURE / Prof. B.G. Shivananda
Principal,
Al-Ameen College of Pharmacy,
Hosur Road, Bangalore -27.
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