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-1-yl)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: 130.131ºC pKa: 15.52 Bioavailability: 56% Half-life: 1 hour

Mechanism of Action: High levels of ATP Cause K+ cannels to close Opening α-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: 222ºC to 226ºC 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 APPROVALIn June 2008, the combination was approved by FDA

MARKET AVAILABILITY

1. Eurepa MF by TORRENT Metformin (500mg), Repaglinide (1mg)Metformin (500mg), Repaglinide (2mg)

2. PrandiMet by Nova NortisMetformin (500mg), Repaglinide (2mg)

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.

NEED OF STUDY

LITERATURE REVIEW REPAGLINIDE:1. 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.

2. UNITED STATES PHARMACOPOEIA:

Liquid chromatography:

25mg to 50ml with methanol

System: 240nm detector

4.6mm x 12.5cm x 5µm packing L

1ml/min flow rate, 45ºC temp, Rt = 1

Mobile phase – methanol and 2.5pH potassium phosphate buffer at 80:20

injection volume = 10µl

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

4. 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

Thermo BDS C18 column 250mm x 4.6mm x 5µ

Mobile phase – phosphate buffer pH=6 : acetonitrile at 45:55

UV detection at 288nm

flow rate 1ml/min at injection 10µl

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 5µm

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 5µm

Excitation and emission wavelengths – 379nm, 282nm

Mobile phase – acetonitrile and 4.5pH ammonium acetate at 70:30

Temperature – 30ºC

Flow rate 1ml/min , Detection – 240nm

8. Quantitative Analysis of Repaglinide in Tablets by RP-TLC with Densitometric UV Detection

Anna Gumieniczek et al Journal of Planar Chromatography-Modern 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 = 0.55±0.03

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 Pharmacopoeia0.1g 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 5µm packing L, temperature = 45ºC

Flow rate = 1ml/min , Rt = 1, Volume = 10µl

3. 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 3µm

Detection – Mass spectrometer (ESI+ )

4. 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 5µm

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 5µm

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 5µm

Mobile Phase- ACN and Methanol 1:1 with Sodium Dodecyl Sulphate 5mM, pH=3.5 with H3PO4

Flow rate = 1ml/min, Temperature = 40ºC , 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 5µm

Mobile phase- ACN and 0.01M Monobasic potassium phosphate buffer pH=7.0 at 20:80, Flow rate 1ml/min

Detection = 236nm, temperature = 30ºC, 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 5µm

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 5µm

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 µg/ml metformin, 10-50 µg/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. Study of physicochemical properties

2. Selection of detector

3. Selection of suitable Stationary phase and Mobile phase

4. Initial method development

5. Optimization of the results obtained

6. 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 & Gilman’s The Manual Of Pharmacology and

Therapeutics - 11th Ed. (2006) pg 1039 – 1060 Lippincott’s Illustrated Reviews : Pharmacology – 3rd edition

pg- 281,289,290 Wilson and Gisvold’s 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 DetectionAnna Gumieniczek et al Journal of Planar

Chromatography-Modern 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 HClG.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)

THANK YOU