“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF SOME ANTIDIABETIC DRUGS”
Synopsis for registration of M.Pharm Dissertation
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
In partial fulfillment
Of the requirement for the Degree of
Master of pharmacy in Pharmaceutical Analysis
Under the Guidance
Professor and Head
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
Department of Pharmaceutical analysis
Karnataka College of Pharmacy, Bengaluru-64
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
PROFORMA FOR REGESTIRATION OF SUBJECTS FOR DISSERTATION1. / Name of the candidate and address / KONDAVITI SAHINI
KARNATAKA COLLEGE OF PHARMACY,
ASWINI HOSPITAL, POST OFFICE ROAD,
KONDAPALLI, IBRAHIMPATNAM (MDL),
2. / Name of the Institution / KARNATAKA COLLEGE OF PHARMACY,
3. / Course of study and subject / MASTER OF PHARMACY
4. / Date of admission / 08-08-2012
5.. / Title of the topic:
“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SOME ANTIDIABETIC DRUGS”
BRIEF RESUME OF THE INTENDED WORK:6.1 Need for the study:
Diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose concentration – hyperglycemia. High blood sugar levels can cause several symptoms, including Blurry vision, Excess thirst, Fatigue, Hunger, Urinating often, Weight loss etc…
Anti-diabetic medicationstreatdiabetes mellitusby loweringglucoselevels in the blood. With the exceptions ofinsulin all are administered orally and are thus also calledoral hypoglycemic agentsororalantihyperglycemic agents. Insulin is administered subcutaneously (by intravenous infusion in emergencies).
On literature survey, it was found that not much work has been done on this particular drug for its determination in bulk and pharmaceutical dosage forms using HPLC and UV techniques. In view of the need for a suitable method for routine analysis, attempts are being made to develop simple, precise and accurate analytical methods to determine title ingredient in bulk and formulation. 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 purport to do. Hence there is a need to validate the new methods developed.
Classification of Antidiabetic drugs:
There are different classes of anti-diabetic drugs, and their selection depends on the nature of the diabetes, age and situation of the person, as well as other factors.
B)Oral hypoglycemic drugs:
- First generation Sulfonylureas
- Second generation Sulfonylureas
- Non Sulfonylureas - Metaglinides
- Alpha glucosidase inhibitors
- Dipeptidyl peptidase - 4
IUPAC name: (S)-1-[2-(3-Hydroxyadamantan-1-ylamino)acetyl]pyrrolidine-2-carbonitrile
Molecular formula: C17H25N3O2
Mechanism of action:
Inhibition of dipeptidyl peptidase-4 (DPP-4) by vildagliptin prevents degradation of glucagon-like peptide-1 (GLP-1) and reduces glycaemia in patients with type 2 diabetes mellitus, with low risk for hypoglycemia and no weight gain. Vildagliptin binds covalently to the catalytic site of DPP-4, eliciting prolonged enzyme inhibition. This raises intact GLP-1 levels, both after meal ingestion and in the fasting state. Vildagliptin has been shown to stimulate insulin secretion and inhibit glucagon secretion in a glucose-dependent manner. At hypoglycemic levels, the counter regulatory glucagon response is enhanced relative to baseline by vildagliptin.
Vildagliptin inhibits hepatic glucose production, mainly through changes in islet hormone secretion, and improves insulin sensitivity, as determined with a variety of methods. These effects underlie the improved glycaemia with low risk for hypoglycemia. Vildagliptin also suppresses postprandial triglyceride (TG)-rich lipoprotein levels after ingestion of a fat-rich meal and reduces fasting lipolysis, suggesting inhibition of fat absorption and reduced TG stores in non-fat tissues.
IUPAC name: p- toluic acid, (+)-2-ethoxy-α-[[(S)-α-isobutyl-o-piperidino-benzyl]carbamoyl].
Molecular formula: C28H28N2O4
Mechanism of Action:
The drug is found to exert its action by stimulating insulin secretion by binding to and inhibiting the ATP-dependent K+ channels in the β-cell membrane, resulting ultimately in an opening of Ca2+ channels. It gets absorbed more or less rapidly and completely from the GI tract and also is exhaustively metabolized in the liver by two biochemical phenomena, such as: (a) glucuronidation; and (b) oxidative biotransformation. Besides, it has been established that the hepatic cytochrome P-450 system 3A4 is predominantly involved in the ultimate metabolism of repaglinide. However, this specific metabolism may be reasonably inhibited by certain drug substances for instance: miconazole, ketoconazole and erythromycin .
IUPAC name: Imidodicarbenimidic diamide,N, N- dimethyl-, monohydrochloride ;
Molecular formula: C4H11N5.HCl
Mechanism of action:
The drug is found to lower both basal and postprandial glucose. Interestingly, its mechanism of action is distinct from that of sulphonylureas and does not cause hypoglycemia. However, it distinctly lowers hepatic glucose production, reduces intestinal absorption of glucose, and ultimately improves insulin sensitivity by enhancing appreciably peripheral glucose uptake and its subsequent utilization. The drug is mostly eliminated unchanged in the urine, and fails to undergo hepatic metabolism .
IUPAC name: (R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine.
Molecular formula: C16H15F6N5O
Mechanism of action:
Sitagliptin works tocompetitively inhibittheenzymedipeptidyl peptidase 4 (DPP-4). This enzyme breaks down theincretinsGLP-1and GIP, gastrointestinal hormonesreleased in response to a meal.By preventing GLP-1 and GIP inactivation, they are able to increase the secretion of insulin and suppress the release of glucagon by the pancreas. This drives blood glucose levels towards normal. As the blood glucose level approaches normal, the amounts of insulin released and glucagon suppressed diminishes, thus tending to prevent an "overshoot" and subsequent low blood sugar (hypoglycemia) which is seen with some other oral hypoglycemic agents.
Sitagliptin has been shown to lowerHbA1clevel by about 0.7% points versus placebo. It is slightly less effective than metformin when used as amonotherapyand does not cause a weight gain compared toSulfonylureas. Sitagliptin is recommended as a second line drug (in combination with other drugs) after the treatment based on a combination of diet and metformin fails. 
IUPAC name: 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3- methyl-1-[(4-methylquinazolin-2-yl)methyl]-3,7-dihydro-1H-purine-2,6-dione.
Molecular formula: C25H26N8O2
Mechanism of action:
Linagliptin is an inhibitor ofDPP-4, an enzyme that degrades the incretin hormonesglucagon-like peptide-1(GLP-1) andglucose-dependent insulinotropic polypeptide(GIP). Both GLP-1 and GIP increase insulin biosynthesis and secretion from pancreatic beta cells in the presence of normal and elevated blood glucose levels. GLP-1 also reducesglucagonsecretion from pancreatic alpha cells, resulting in a reduction in hepatic glucose output. Thus, linagliptin stimulates the release of insulin in a glucose-dependent manner and decreases the levels of glucagon in the circulation.
IUPAC name: 1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulphonyl]-3(trans-4-methylcyclohexyl) urea.
Molecular formula: C24H34N4O5S.
Mechanism of action:
The drug is found to be metabolized primarily through oxidation of the alkyl side chain attached to the pyrrolidine nucleus via a minor metabolic path that essentially involves acetylation of the amine function .
6.2 REVIEW OF LITERATURE:
- Maha Farouk et al., reported on Validated spectrophotometric methods for determination of some oral hypoglycemic drugs.
- Samina A. Jamadar et al., reported on Development and validation of UV spectrophotometric method for the determination of Gliclazide in tablet dosage form.
- Karthik A et al., reported on simultaneous determination of pioglitazone and glimepiride in bulk drug and pharmaceutical dosage form by RP-HPLC method.
- Ayyapan. J et al., reported on validated HPLC method for the fast and sensitive determination of few antidiabetic drugs residues in support of cleaning validation in formulation area.
- Dalia Rashad El-Wasseef reported on Simultaneous Determination of Metformin, Nateglinide and Gliclazide in Pharmaceutical Preparations Using Micellar liquid Chromatography.
- H. H. Gadape and K. S. Parikh reported on Quantitative determination and Validation of Pioglitazone in Pharmaceutical using Quantitative Nuclear Magnetic Resonance Spectroscopy. 
- Shrotri C.K reported on a simple electroanalytical method for estimation Rosiglitazone maleate and Metformin hydrochloride individually from pharmaceutical formulation.
- Madhukar. A et al., reported on rapid and sensitive RP-HPLC analytical method development and validation of Pioglitazone hydrochloride.
- Gadapa nirupa et al., reported on RP-HPLC analytical method development and validation for simultaneous estimation of three drugs: Glimepiride, Pioglitazone, and Metformin and its pharmaceutical dosage forms.
- M.S.V.Sakuntala et al., reported on a RP-HPLC method development and validation for the simultaneous estimation of Glimepiride and Pioglitazone hydrochloride in tablet dosage forms.
- Najam sultana et al., reported on Development and Validation of New Assay Method for the Simultaneous Analysis of Diltiazem, Metformin,Pioglitazone and Rosiglitazone by RP-HPLC and its Applications in Pharmaceuticals and Human Serum.
- Nahed M El-Enany et al., reported on Development and validation of a rephrased phase- HPLC method for simultaneous determination of rosiglitazone and glimepiride in combined dosage forms and human plasma.
- Hapse S. A et al., reported onSimultaneous Estimation of Metformin and Glimepiride by Ultraviolet Spectrophotometry.
- SurendraBabu B et al., reported onMethod development and validation of simultaneous estimation of metformin and pioglitazone in bulk and pharmaceutical formulation by UPLC.
- Freddy H.Havaldar et al., reported on Simultaneous Estimation of Glimepiride,Rosiglitazone and Pioglitazone Hydrochloridein the Pharmaceutical Dosage Form.
- Yuni Retnaningtyas et al., reported on Development and validation of TLC densitometry method for the determination of Gliclazide in tablet dosage form.
- Harshal Kanubhai Trivedi et al., reported on development and validation of an UPLC method for in-vitro study of Glipizide extended release tablets.
- N.Monila et al., reported on New Extractive Method Development of Sitagliptin Phosphate in API and Its Unit Dosage Forms by Spectrophotometry.
- Rudy Bonfilio et al., reported onDevelopment and Validation of an UV-Derivative Spectrophotometric Method for Determination of Glimepiride in Tablets.
- Amruta B. Loni et al., reported on Simultaneous UV Spectrophotometric Method for Estimation of Sitagliptin phosphate and Metformin hydrochloride in Bulk and Tablet Dosage Form.
- Patil Sudarshan S et al., reported on Development and Validation of analytical method for Simultaneous Estimation of Glibenclamide and Metformin HCl in Bulk and Tablets using UV – visible spectroscopy.
- M.Sarat et al., reported on RP-HPLC method for simultaneous estimation of Sexagliptin and Pioglitazone in tablets.
- Cornelius Hess et al., reported on simultaneous identification and validated quantification of 11oral hypoglycemic drugs in plasma by electrospray ionization liquid chromatography- mass spectrometry.
- Mohammad Abdul‐Azim Mohammad et al., reported on Development and validation of a reversed‐phase column liquid chromatographic method for simultaneous determination of two novel gliptins in their binary mixtures with Metformin .
The study makes an attempt to establish sensitive and accurate methods for estimation of antidiabetic drugs in pharmaceutical dosage form:
- To develop a new sensitive and accurate chemometric based techniques and also spectroscopic methods for estimation antidiabetic drugs in pharmaceutical dosage form.
- To develop a new sensitive and accurate HPLC method.
- Validating the proposed newly developed methods in accordance with the analytical parameters mentioned in the IP, USP, BP and ICH guidelines.
- To apply the proposed newly developed methods for analysis of these drugs in their bulk and pharmaceutical dosage form.
7.1 Source of data:
- Literature survey was done at Karnataka College of Pharmacy using internet facilities (RGUHS HELINET).
- References from library at RGUHS, Bengaluru.
- Indian Instituteof Sciences online library,Bengaluru.
In the present investigation of the new analytical spectroscopic methods for the estimation of some antidiabetic drugs is in need and HPLC with different solvent system and other chromatographic instruments.
The entire instruments like UV-Spectrophotometer(Shimadzu 1700), HPLC (agilent 1120 binary, gradient) etc are available. Analytical method and development on some anti-diabetic drugs shall be carried out at Department of Pharmaceutical Analysis in Karnataka College of Pharmacy, Bengaluru. This lab is adequately equipped with necessary analytical setup
to carry out desired work.
For the selection of mobile phases to be used in HPLC,different combinations and ratio of solvent will be tried to obtain good retention time and chromatogram having optimum resolution. Various data collected for the analytical method development either by spctrophotometry or by HPLC will be treated statisticallyto find its compliance with ICH or pharmacopoeial guidelines.Typical analytical parameters such as linearity, range, precision, specificity, accuracy, ruggedness, and robustness used in assay validation will be determined.
- International journal of pharmaceutical Research and Analysis.
- International current pharmaceutical journal
- Indian journal of Medical Research.
- International journal of pharmaceutical sciences and research
- IOSR Journal of Pharmacy and Biological Sciences
- International journal of pharmaceutical Research and development
- International journal of pharmacy and pharmaceutical sciences.
- International journal of Biomedical sciences
- International Research Journal of Pharmaceutical and Applied Sciences
- Journal of Chromatographic science
- Drug discovery and therapeutics journal.
- Journal of chemical and pharmaceutical research
- Der Pharmacia Lettre
- Journal of chemistry
- Chemistry central journal
- Current pharma research
- E- Journal of chemistry.
7.3 Does the study require any investigation or intervention to be conducted on
Patients or other humans or animals?
7.4 Has ethical clearance been obtained from your institution in case of 7.4?
- Rang and Dale’s pharmacology text book, seventh edition; Elsevier Churchill Livingston publishers 2007: 372 – 84.
- Medicinal chemistry by Ashutosh Kar, new age international(p) limited publishers; third edition 2005: 584 – 99.
- Herman G, Bergman A, Liu F, Stevens C, Wang A, Zeng W, Chen L, Snyder K, Hilliard D, Tanen M, Tanaka W, Meehan A, Lasseter K, Dilzer S, Blum R, Wagner J. "Pharmacokinetics and pharmacodynamic effects of the oral DPP-4 inhibitor Sitagliptin in middle-aged obese subjects."J ClinPharmacol 2006;46(8): 876–86.
- Balas B, Baig MR, Watson C, Dunning BE, Ligueros-Saylan M, Wang Y, He YL, Darland C, Holst JJ, Deacon CF, Cusi K, Mari A, Foley JE, DeFronzo RA: The dipeptidyl peptidase IV inhibitor vildagliptin suppresses endogenous glucose production and enhances islet function after single-dose administration in type 2 diabetic patients. J Clin Endocrinol Metab. 2007 Apr;92(4):1249-55.
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- Maha Farouk, Osama Abdel-Satar, Omar Abdel-Aziz, Maya Shaaban. Validated spectrophotometric methods for determination of some oral hypoglycemic drugs. Drug Discoveries & Therapeutics. 2011; 5(1):41-52.
- Samina A. Jamadar, Snehal P. Mulye, Poonam S. Karekar , Yogesh V. Pore, Kishor B. Burade. Development and validation of UV spectrophotometric method for the determination of Gliclazide in tablet dosage form.Der Pharma Chemica 2011; 3 (4):338-343.
- Karthik A, Subramanian G, Mallikarjuna Rao C, Krishnamurthy bhat, Ranjithkumar A, Musmad P, Surulivelrajan M, Karthikayan K and Udapa N. Simultaneous determination of pioglitazone and glimepiride in bulk drug and pharmaceutical dosage form by RP-HPLC method. Pak. J. Pharm. Sci 2008; 21(4):421-425.
- Ayyapan. J, Umapathi. P, Darlin Quine. S. validated HPLC method for the fast and sensitive determination of few antidiabetic drugs residues in support of cleaning validation in formulation area. International Journal of Pharmacy and Pharmaceutical Sciences 2011; 3(5):0975-1491.
- Dalia Rashad El-Wasseef. Simultaneous Determination of Metformin, Nateglinide and Gliclazide in Pharmaceutical Preparations Using Micellar Liquid Chromatography.International Journal of Biomedical Science 2012;8(2): 144-151.
- H. H. Gadape and K. S. Parikh. Quantitative determination and Validation of Pioglitazone in Pharmaceutical using Quantitative Nuclear Magnetic Resonance Spectroscopy.J. Chem. Pharm. Res 2011; 3(1):649-664.
- Shrotri .C.K, Sathe.P.A, Rege.P.V. Simple electroanalytical method for estimation Rosiglitazone maleate and Metformin hydrochloride individually from pharmaceutical formulation. IJPRD2011;Vol 3(12): 64-71.
- Madhukar. A1, K. Naresh, CH. Naveen Kumar, N. Sandhya1and P. Prasanna. Rapid and sensitive RP-HPLC analytical method development and validation of Pioglitazone hydrochloride. Der Pharmacia Lettre 2011; 3(3): 128-132.
- Gadapa Nirupa and Upendra M.Tirpathi. RP-HPLC analytical method development and validation for simultaneous estimation of three drugs: Glimepiride, Pioglitazone, and Metformin and its pharmaceutical dosage forms. Journal of Chemistry volume 2013.
- M. S. V. Sakuntala, S. V. U. M. Prasad, S. Sri Devi, S. Kishore Yadav, K. Srinivas Reddy.A RP- HPLC method development and validation for the simultaneous estimation of glimepiride and pioglitazone HCl in tablet dosage forms. Journal of Chemical and Pharmaceutical Research 2012; 4(1):154-159.
- Najma Sultana, M. Saeed Arayne, Nighat Shafi, Farhan Ahmed Siddiqui, and Azhar Hussain. Development and Validation of New Assay Method for the Simultaneous Analysis of Diltiazem, Metformin,Pioglitazone and Rosiglitazone by RP-HPLC and its Applications in Pharmaceuticals and Human Serum. Journal of Chromatographic Science 2011;49.
- Nahed M El-Enany, Amina A Abdelal, Fathalla F Belal, Yoshinori I Itoh and Mitsuhiro N Nakamura. Development and validation of a rephrased phase- HPLC method for simultaneous determination of rosiglitazone and glimepiride in combined dosage forms and human plasma. El-Enany et al. Chemistry Central Journal 2012; 6:9.
- Hapse S. A., Suruse S.D., Ugale S.S., Magar S.D.Simultaneous Estimation of Metformin and Glimepiride by Ultraviolet Spectrophotometry. CPR 2012;2(3): 557-559.
- Surendra Babu B, Sravani, Ravi Kumar.Method development and validation of simultaneous estimation of metformin and pioglitazone in bulk and pharmaceutical formulation by UPLC.Int. Res J Pharm. App Sci 2012; 2(4): 91-96.
- Freddy H. Havaldar and Dharmendra L.Vairal.Simultaneous Estimation of Glimepiride,Rosiglitazone and Pioglitazone Hydrochloride in the Pharmaceutical Dosage Form.E-Journal of Chemistry 2010;7(4):1326-1333.
- Yuni Retnaningtyas, Lestyo Wulandari, Feby Erliana.Development and validation of TLC densitometry method for the determination of Gliclazide in tablet dosage form. International Current Pharmaceutical Journal 2012; 1(10): 332-335.
- Harshal Kanubhai Trivedi, Nayan Kshtri, Vipul Patel and Vinay Roa. On development and validation of an UPLC method for in-vitro study of Glipizide extended release tablets. IJPSR 2012; 3(9): 3317-3322.
- N.Monila1, Ravi Pratap Pulla1, Harshini Shabad1, V.Swathi1, J.Rajasekhar1, A.Ramesh1, B.Koti Reddy2 & B.Umashankar. New Extractive Method Development of Sitagliptin Phosphate in API and Its Unit Dosage Forms by Spectrophotometry.ISSN 2012; 1(16):37-40.
- Rudy Bonfilio, Magali B. de Araujob and Herida R. N. Salgadoa.Development and Validation of an UV-Derivative Spectrophotometric Method for Determination of Glimepiride in Tablets.J. Braz. Chem. Soc2011;22(2):292-299.
- Amruta B. Loni, Minal R. Ghante, S. D. Sawant.Simultaneous UV Spectrophotometric Method for Estimation of Sitagliptin phosphate and Metformin hydrochloride in Bulk and Tablet Dosage Form.Der Pharma Chemica, 2012; 4 (3): 854-859.
- PatilSudarshan S, Bonde C. G.Development and Validation of analytical method for Simultaneous Estimation of Glibenclamide and Metformin HCl in Bulk and Tablets using UV – visible spectroscopy. International Journal of ChemTech Research 2009;1(4):905-909
- M.Sarat, P.Murali Krishna, and C.Rambabu. RP-HPLC method for simultaneous estimation of Sexagliptin and Pioglitazone in tablets. IRJP 2012; 3(5).
- Cornelius Hess, Frank Musshoff, Burkhard Madea. Simultaneous identification and validated quantification of 11 oral hypoglycemic drugs in plasma by electrospray ionization liquid chromatography- mass spectrometry. Anal Bioanal Chem2011; 400:33-41.
- Mohammad Abdul‐Azim Mohammad, Ehab Farouk Elkady and Marwa Ahmed Fouad. Development and validation of a reversed‐phase column liquid chromatographic method for simultaneous determination of two novel gliptins in their binary mixtures with Metformin. European Journal of Chemistry 2012;3(2):152‐155.
9 / Signature of the candidate / [K.SAHINI]
10 / Remarks of the guide: The topic selected for dissertation is satisfactory. Adequate equipment and chemicals are available to carry out the project work.
11 / Name and Designation [BLOCK LETTERS]
11.1 / Guide / Dr.C.SREEDHAR,
PROFESSOR AND HEAD,
DEPARTMENT OF PHARMACEUTICAL ANALYSIS,
KARNATAKA COLLEGE OF PHARMACY,
11.2 / Signature of Guide / [Dr.C.SREEDHAR]
11.3 / Co-Guide
11.4 / Signature of Co-Guide
11.5 / Head of the Department / Dr.C.SREEDHAR,
PROFESSOR & HEAD,
DEPT OF PHARMACEUTICAL ANALYSIS,
KARNATAKA COLLEGE OF PHARMACY,
11.6 / Signature of HOD / [Dr.C.SREEDHAR]
12 / 12.1 / Remarks of the Principal:All the required facilities will be provided to carry out dissertation work under the supervision of guide
12.2 / Principal / Dr.K.RAMESH,
KARNATAKA COLLEGE OF PHARMACY,
HEGDE NAGAR MAIN ROAD,
YELAHANKA, HOBLI, BANGLORE.
12.3 / Signature of the principal / [Dr.K.RAMESH]