SIMULTANEOUS ESTIMATION OF ANTIDIABETICS METFORMIN HYDROCHLORIDE AND REPAGLINIDE USING RP-HPLC

BY CHINTALAPATI SUJITHA 11DQ1S0404 PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE JOGI NPALLY BR PHARMACY COLLEGE

CONTENTS:
INTRODUCTION DRUG PROFILE NEED OF STUDY LITERATURE REVIEW AIM AND OBJECTIVE PLAN OF WORK BIBLIOGRAPHY

INTRODUCTION

Analytical chemistry may be defined as the science and art of determining the composition of materials in terms of the elements or compounds contained Analytical method development is the process of developing a new method of analysis that is simple, precise, accurate and reliable. Analytical instrumentation plays an important role in the production and evaluation of new products The instrumentation provides lower detection limits required to assure safety

DIFFERENT ANALYTICAL TECHNIQUES AVAILABLE

I.

Spectrometric techniques:
Atomic spectrometry (Emission and Absorption) Electron Spin Resonance Spectroscopy Fluorescence and Phosphorescence Spectrophotometry Infrared Spectrophotometry Nuclear Magnetic Resonance Spectroscopy Radiochemical Techniques Raman Spectroscopy Ultraviolet and Visible Spectrophotometry X-Ray Spectroscopy

II.

Chromatographic Techniques
Gas Chromatography High Performance Liquid Chromatography Thin Layer Chromatography

III.

Electro Analytical Methods

Amperometry (Electrical Current) Potentiometry (Electric Potential) Conductometry (Electrical conduction) Voltammetry (Electrical Voltage) Electro gravimety (Electric Potential)

IV.

Miscellaneous Techniques
Kinetic Techniques Mass Spectrometry Thermal Analysis

V.

Hyphenated Techniques
GC-MS ICP-MS GC-IR MS-MS LC-MS

DRUG PROFILE

REPAGLINIDE:

Chemically (S)-(+)-2-ethoxy-4-[2-(3-methyl-1-[2-(piperidin-1yl)phenyl]butyl amino)-2-oxoethyl]benzoic acid Molecular weight: 452.59

Solubility : Practically insoluble in water, freely soluble in methanol and methylene chloride Nature: White or almost white powder Melting point: 130.131C pKa: 15.52 Bioavailability: 56% Half-life: 1 hour Mechanism of Action: High levels of ATP Cause K+ cannels to close Opening -type Ca2+ channels Repaglinide inhibits K+ channels

Therapeutic use: Used as an adjunct to diet and exercise to lower blood glucose in patients with noninsulin dependent diabetic mellitus whose hyperglycemia cannot be controlled by diet and exercise alone. Can be used with metformin or thiazolidine diones
Trade Names: Prandin- 0.5mg, 1mg, 2mg tablets by Novo Nortis glucoNorm- in Canada

METFORMIN

Metformin HCl (N,N-dimethylimidodicarbonimidic diamide hydrochloride)

Molecular weight : 165.6

Solubility: Freely soluble in water, Slightly soluble in alcohol, insoluble in acetone and methylene chloride Nature: white or almost white crystals Melting point: 222C to 226C pKa: 12.4 Bioavailability: 50% to 60% absolute bioavailability Half life: 3 to 6hrs Mechanism of Action:

Decreases hepatic glucose production Decreases intestinal absorption of glucose Improves insulin sensitivity by increasing peripheral glucose uptake and utilization

METFORMIN

AMPK

GLUT4 DEPLOYMENT

INSULIN DEPENDENT GLUCOSE UPTAKE

AMPK plays important role in insulin signaling, whole body energy balance and metabolism of glucose and fats AMPK activity in skeletal muscles also increased

Therapeutic use: Improves glucose tolerance in patients with NIDDM lowering both basal and post prandial plasma glucose

TRADE NAMES

Glucophage 500mg, 850mg, 1g Glucophage XR- 500mg, 750mg Glucometza- 500mg, 1g Fortamet- 500mg, 1g Riomet oral solution by Ranbaxy 500mg/5ml

METFORMIN AND REPAGLINIDE COMBINATION

Repaglinide/metformin combination therapy is indicated as an adjunct to diet and exercise to improve glycemic control in adults with T2DM who are already treated with a meglitinide and metformin or who have inadequate glycemic control on monotherapy with meglitinide or metformin. A single combination tablet offers increased patient convenience and the potential for increased adherence to therapy. The rationale for this combination is based on the premise that both repaglinide and metformin target different pathophysiological components of T2DM (insulin secretion and insulin resistance, respectively) and different aspects of hyperglycemia (postprandial and fasting, respectively). In non obese T2DM patients, metformin reduced postprandial glycemia, triglycerides and free fatty acids similarly to repaglinide. These data support a possible synergistic effect on postprandial hyperglycemia by combining metformin and repaglinide.

FDA APPROVAL
In June 2008, the combination was approved by FDA MARKET AVAILABILITY

1.

Eurepa MF by TORRENT Metformin (500mg), Repaglinide (1mg) Metformin (500mg), Repaglinide (2mg) PrandiMet by Nova Nortis
Metformin (500mg), Repaglinide (2mg)

2.

NEED OF STUDY

The drug or drug combination may not be official in any pharmacopoeias, A proper analytical procedure for the drug may not be available in the literature due to patent regulations, Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excepients, Analytical methods for the quantitation of the drug in biological fluids may not be available, Analytical methods for a drug in combination with other drugs may not be available, The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.

LITERATURE REVIEW

1.

REPAGLINIDE:
BRITISH PHARMACOPOEIA: Potentiometry: 0.320g in 10ml methanol R and add 60ml of anhydrous acetic acid R. Titrate with 0.1M Perchloric acid, determining end point potentiometrically. UNITED STATES PHARMACOPOEIA: Liquid chromatography: 25mg to 50ml with methanol System: 240nm detector 4.6mm x 12.5cm x 5m packing L 1ml/min flow rate, 45C temp, Rt = 1 Mobile phase methanol and 2.5pH potassium phosphate buffer at 80:20 injection volume = 10l

2.

3.

Determination of Repaglinide in Pharmaceutical Formulations by HPLC with UV Detection

M.Gandhimathi et al Analytical Sciences Dec 2003 Vol 9 (1675) Shim-pack, RP-C18 Column 150mm x 4.6mm Mobile phase- Methanol:0.1% v/v triethlyamine (pH adjusted to 7 with orthophosphoric acid) at 50 :50 Detection at 235nm Flow rate 1ml/min

High Performance Liquid Chromatographic Method for Estimation of Repaglinide in Tablet Dosage Forms
4.

Meeta.A.Jiladia et al International Journal of Pharma and Bio Sciences V1(2)2010 Thermo BDS C18 column 250mm x 4.6mm x 5 Mobile phase phosphate buffer pH=6 : acetonitrile at 45:55 UV detection at 288nm flow rate 1ml/min at injection 10l

5.

Determination of Repaglinide in Pharmaceutical Formulations by RP-HPLC method

Prameela Rani et al Journal of Applied Sciences Research 1500-1504, 2009 Column- C18 250mm x 4.6mm x 5m Mobile phase Methanol and Ammonium Phosphate buffer (pH = 2.5) in 70 : 30, flow rate = 1ml/min Run time = 12min, Detection = 245

6.

Determination of Repaglinide and the Related Substances in Repaglinide and its Tablets by HPLC
Sun Hao et al China Pharmacy 2007-19 Kromasil C18 column Mobile phase 0.05mol/l acetate buffer (pH=4) & methanol (20:80) Detection 243nm, flow rate 1ml/min

7.

Determination of Spectrofluorometric and HPLC methods for Invitro Analysis of Repaglinide

A.K.Tiwari et al Indian J Pharm Sci Mar-apr, 72(2):240-244 Column 250mm x 4.6mm x 5m Excitation and emission wavelengths 379nm, 282nm Mobile phase acetonitrile and 4.5pH ammonium acetate at 70:30 Temperature 30C Flow rate 1ml/min , Detection 240nm

8.

Quantitative Analysis of Repaglinide in Tablets by RPTLC with Densitometric UV Detection

Anna Gumieniczek et al Journal of Planar ChromatographyModern TLC (1789-0993) RP-8 TLC plates Mobile phase- Acetonitrile and pH 6 phosphate buffer 60 : 40 Wave length 225nm

9.

Estimation of Repaglinide in Bulk and Tablet Dosage Forms by HPTLC

Meeta.A.Jiladia et al International Journal of Pharmacy and Pharmaceutical Sciences vol.1 sup.1 Nov-2009 Silica gel 60 F254 TLC plate Mobile phase Chloroform, methanol, ammonia at 4.5:0.8:0.05 v/v Rf = 0.550.03 Scanned at 288nm

10.

Electrochemical Determination of Antidiabetic drug Repaglinide

Mohammed Abdel Nabi EL-RIES et al The Pharmaceutical Society of Japan Britton Robinson Buffer Carbon paste and Glassy Carbon Electrodes Well defined peak at pH 2 to 11

METFORMIN
1.

British Pharmacopoeia
0.1g of Metformin HCl added with 70ml water for 15min. Dilute to 100ml with water and filter discarding first 20ml Dilute 10ml filtrate to 100ml with water and 10ml resulting solution to 100ml with water. Absorbance at 232nm, A1%-1cm = 798

2.

United States Pharmacopoeia

Mobile phase methanol and potassium phosphate buffer pH 2.5 at 80:20 25mg accurately weighed to 50ml flask with methanol column 12.5cm x 4.6mm x 5m packing L, temperature = 45C Flow rate = 1ml/min , Rt = 1, Volume = 10l

3.

4.

Simple and Rapid Method Determination for Metformin in Human Plasma using High Performance Liquid Chromatography Tandem Mass Spectrometry : Application to Pharmacokinetic Studies Eduardo Werneck-Barroso et al Journal of Chromatography B, 852(2007) 308-316 Plasma deproteinization using Acetonitrile Mobile phase- Water, ACN, Formic acid in 55:45:0.048%v/v Run time = 3min column C18 Pursuit 100mm x 20mm x 3m Detection Mass spectrometer (ESI+ ) HPLC Method for Estimation of Metformin HCl in Formulated Microspheres and Tablet Dosage Forms
Column- RP Phenomex C18 Column Mobile phase- Acetonitrile, pH 5.75 phosphate buffer at 65:35

5.

Simultaneous Determination of Metformin and Pioglitazone Tablets in Pharmaceutical Dosage form by RP-HPLC Method
Srinivas.P et al International Journal of Pharmaceutical, Chemical and Biological Sciences (2249-5504) Column- Zorbax XDB 4.6mm x 150mm x 5m Mobile phase- Disodium Hydrogen Phosphate, Acetonitrile, Triethylamine pH to 7.10 in 660:340:1 Flow rate 1ml/min, Detection 225nm, Rt = 3.2, 7.3

6.

Validated Method for Simultaneous Estimation of Metformin HCl and Vildagliptin by RP-HPLC in Bulk and Pharmaceutical Dosage Forms
B.Santosha et al International Journal of Pharmaceutical and Applied Sciences (2277-4149) Column- Dionex C18 250mm x 4.6mm x 5m Dipotassium hyd phosphate buffer(0.01m), water at 90:10, 1.5ml/min Detection 215nm, Rt = 2.390, 4.601

7.

Validation of a HPLC Method for Simultaneous Analysis of Metformin and Gliclazide in Human Plasma
Maria-Cristina Ranetti et al Farmacia 2009, vol. 57, 6 Zorbax Eclipse C18 XDB 150mm x 4.6mm x 5m Mobile Phase- ACN and Methanol 1:1 with Sodium Dodecyl Sulphate 5mM, pH=3.5 with H3PO4 Flow rate = 1ml/min, Temperature = 40C , Detection = 236nm

8.

Estimation and Pharmacokinetics of Metformin in Human Volunteers

D.Bhavesh et al Indian J. Pharm. Educ. And Research Cation exchange column Hih Chrom, 250mm x 4.6mm Mobile phase Methanol and Potassium dihydrogen orthophosphate buffer pH=3.5 46:54%v/v Flow rate=1ml/min , Rt=13min, Detection = 236nm

9.

Analysis of Metformin in Ante mortem Serum and Postmortem Specimens by a Novel HPLC Method and Application to an Intoxication Case
Karla.A.Moore et al Journal of Analytical Toxicology vol.27 Column-Phenomex Prodigy Rp column 150mm x 4.6mm x 5m Mobile phase- ACN and 0.01M Monobasic potassium phosphate buffer pH=7.0 at 20:80, Flow rate 1ml/min Detection = 236nm, temperature = 30C, Rt=6.3min

10.

Spectrophotometric Analysis of Metformin HCl

G.Mubeen et al. Colorimetric complex formation by NaOH and Ninhydrin Detection at 570nm

METFORMIN AND REPAGLINIDE COMBINATION

1.

Determination and Validation of RP-HPLC methods for Simultaneous Estimation of Metformin HCl and Repaglinide in Tablet Dosage Form
Madhavi Jain et al International Journal of Pharmaceutical and Research Sciences (2278-9464) Column-Chromatopak C18 250mm x 4.6mm x 5m Mobile phase- Acetonitrile and water at 90:10 Flow rate 1ml/min, Detection = 223nm, Run time = 10min

2.

Determination of RP-HPLC method for Metformin and Repaglinide in Rabbit Plasma

T.Ramanji Reddy et al International Journal of Pharmacy and Pharmaceutical Sciences (0975-1491) Solvent extraction by ethyl acetate and 0.1N HCl Column- C18 150mm x 4.6mm x 5m Mobile phase- ACN and pH=4 Phosphate buffer at 60:40 with 1% TEA, Flow rate 0.8ml/min Detection 254nm, Gradient elution technique

3.

Simultaneous Determination of Metformin HCl and Repaglinide in Pharmaceutical Dosage Form by UV Difference Spectrophotometric Method

Mahipal.M.Makwana et al International Journal of Institutional Pharmacy and Life Sciences (2249-6807) Metformin 234.5nm in 0.1N NaOH vs 0.1N HCl Repaglinide 269nm and 304nm in 0.1N NaOH vs 0.1N HCl Linearity 2 12 g/ml metformin, 10-50 g/ml Repaglinide

AIM AND OBJECTIVE

India has more diabetics than any other country in the world, according to the International Diabetes Foundation, although more recent data suggest that China has even more. The disease affects more than 50 million Indians - 7.1% of the nation's adults - and kills about 1 million Indians a year. The average age on onset is 42.5 years. The high incidence is attributed to a combination of genetic susceptibility plus adoption of a high-calorie, low-activity lifestyle by India's growing middle class. The Repaglinide and Metformin combination has wider therapeutic uses but the methods of analysis are very limited More over no official method has been developed for the combination in any of the official pharmacopoeias till date My main objective is to develop a simple, precise, accurate method that is a faster method and can be used for regular analysis

PLAN OF WORK
1. 2.

3.
4. 5. 6.

Study of physicochemical properties Selection of detector Selection of suitable Stationary phase and Mobile phase Initial method development Optimization of the results obtained Validation of the method according to valid ICH guidelines

REFERENCES

The British Pharmacopoeia - 2009, Volume 1 & 2 pages 3813, 5176 The United States Pharmacopoeia - 2007, Indian Pharmacopoeia 2007, Volume 2 page 740 HPLC Method Development By Lloyd R. Snyder, Joseph L. Glajch, J. J. Kirkland Goodman & Gilmans The Manual Of Pharmacology and Therapeutics - 11th Ed. (2006) pg 1039 1060 Lippincotts Illustrated Reviews : Pharmacology 3rd edition pg- 281,289,290 Wilson and Gisvolds Textbook of Organic, Medicinal and Pharmaceutical Chemistry pg 672, 671 Drug Bank DB00331, DB00912

Determination of Repaglinide in Pharmaceutical Formulations by HPLC with UV Detection

M.Gandhimathi et al Analytical Sciences Dec 2003 Vol 9 (1675)

High Performance Liquid Chromatographic Method for Estimation of Repaglinide in Tablet Dosage Forms
Meeta.A.Jiladia et al International Journal of Pharma and Bio Sciences V1(2)2010

Determination of Repaglinide in Pharmaceutical Formulations by RP-HPLC method

Prameela Rani et al Journal of Applied Sciences Research 1500-1504, 2009

Determination of Repaglinide and the Related Substances in Repaglinide and its Tablets by HPLC
Sun Hao et al China Pharmacy 2007-19

Determination of Spectrofluorometric and HPLC methods for Invitro Analysis of Repaglinide A.K.Tiwari et al Indian J Pharm Sci Mar-apr, 72(2):240-244 Quantitative Analysis of Repaglinide in Tablets by RP-TLC with Densitometric UV Detection
Anna Gumieniczek et al Journal of Planar ChromatographyModern TLC (1789-0993)

Estimation of Repaglinide in Bulk and Tablet Dosage Forms by HPTLC

Meeta.A.Jiladia et al International Journal of Pharmacy and Pharmaceutical Sciences vol.1 sup.1 Nov-2009

Electrochemical Determination of Antidiabetic drug Repaglinide

Mohammed Abdel Nabi EL-RIES et al The Pharmaceutical Society of Japan

Simple and Rapid Method Determination for Metformin in Human Plasma using High Performance Liquid Chromatography Tandem Mass Spectrometry : Application to Pharmacokinetic Studies
Eduardo Werneck-Barroso et al Journal of Chromatography B, 852(2007) 308-316

HPLC Method for Estimation of Metformin HCl in Formulated Microspheres and Tablet Dosage Forms Simultaneous Determination of Metformin and Pioglitazone Tablets in Pharmaceutical Dosage form by RP-HPLC Method
Srinivas.P et al International Journal of Pharmaceutical, Chemical and Biological Sciences (2249-5504)

Validated Method for Simultaneous Estimation of Metformin HCl and Vildagliptin by RP-HPLC in Bulk and Pharmaceutical Dosage Forms
B.Santosha et al International Journal of Pharmaceutical and Applied Sciences (2277-4149)

Validation of a HPLC Method for Simultaneous Analysis of Metformin and Gliclazide in Human Plasma
Maria-Cristina Ranetti et al Farmacia 2009, vol. 57, 6

Estimation and Pharmacokinetics of Metformin in Human Volunteers

D.Bhavesh et al Indian J. Pharm. Educ. And Research

Analysis of Metformin in Ante mortem Serum and Postmortem Specimens by a Novel HPLC Method and Application to an Intoxication Case
Karla.A.Moore et al Journal of Analytical Toxicology vol.27

Spectrophotometric Analysis of Metformin HCl

G.Mubeen et al.

Determination and Validation of RP-HPLC methods for Simultaneous Estimation of Metformin HCl and Repaglinide in Tablet Dosage Form
Madhavi Jain et al International Journal of Pharmaceutical and Research Sciences (2278-9464)

Determination of RP-HPLC method for Metformin and Repaglinide in Rabbit Plasma

T.Ramanji Reddy et al International Journal of Pharmacy and Pharmaceutical Sciences (0975-1491)

Simultaneous Determination of Metformin HCl and Repaglinide in Pharmaceutical Dosage Form by UV Difference Spectrophotometric Method
Mahipal.M.Makwana et al International Journal of Institutional Pharmacy and Life Sciences (2249-6807)

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