development and validation of rp-hplc method ......3. faster separation times (minutes) 4....

www.wjpps.com Vol 8, Issue 9, 2019.

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Illendula et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

THE SIMULTANEOUS ESTIMATION OF METFORMIN AND

NATEGLINIDE IN BULK AND COMBINED TABLET DOSAGE FORM

Santhosh Illendula*, M. Sushma, V. Shirisha, Dr. K.N.V. Rao, Dr. Rajeswar Dutt

Department of Pharmaceutical Analysis, Nalanda College of Pharmacy, Cherlapally(v),

Nalgonda(Dt), Telangana (St), India, 508001.

INTRODUCTION

Analysis may be defined as the science and art of determining the

composition of materials in terms of the elements or compounds

contained in them. In fact, analytical chemistry is the science of

chemical identification and determination of the composition (atomic,

molecular) of substances, materials and their chemical structure.

1. The drug or drug combination may not be official in any

pharmacopoeias.

2. A proper analytical procedure for the drug may not be available in

the literature due to Patent regulations.

3. Analytical methods for a drug in combination with other drugs may

not be available.

4. Analytical methods for the quantitation of the drug in biological

fluids may not be available.

5. 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.[1,2]

1.1 DIFFERENT METHODS OF ANALYSIS

The following techniques are available for separation and analysis of components of interest.

Spectral methods

The spectral techniques are used to measure electromagnetic radiation which is either

absorbed or emitted by the sample.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 8, Issue 10, 880-903 Research Article ISSN 2278 – 4357

*Corresponding Author

Santhosh Illendula

Department of

Pharmaceutical Analysis,

Nalanda College of

Pharmacy, Cherlapally(v),

Nalgonda(Dt), Telangana

(St), India, 508001.

Article Received on

06 August 2019,

Revised on 27 August 2019,

Accepted on 17 Sept. 2019

DOI: 10.20959/wjpps201910-14828


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E.g. UV-Visible spectroscopy, IR spectroscopy, NMR, ESR spectroscopy, Flame

photometry, Fluorimetry.2

Electro analytical methods

Electro analytical methods involved in the measurement of current voltage or resistanceas a

property of concentration of the component in solution mixture.

E.g. Potentiometry, Conductometry, Amperometry.[2]

Chromatographic Methods

E.g. Paper chromatography, thin layer chromatography (TLC), High performance thin layer

chromatography (HPTLC), High performance liquid chromatography (HPLC), Gas

chromatography (GC).[2]

Table 1.1: Summary of Hyphenated separation techniques.[2]

Separation Technique Hyphenated mode

Liquid chromatography

Liquid chromatography-mass spectrometry(LC/MS)

Liquid chromatography-Fourier-transform infrared

Spectrometry(LC-FTIR)

Liquid chromatography-nuclear magnetic resonance

spectroscopy(LC/NMR)

Liquid chromatography-inductively coupled plasma mass

spectrometry(LC-ICPMS)

Gas chromatography

Gas chromatography-mass spectrometry(GC/MS)

Gas chromatography-Fourier-transform infrared(GC-

FTIR)

Gas chromatography-FTIR-MS(GC-FTIR-MS)

Capillary electrophoresis

Capillary electrophoresis-mass spectrometry(CE/MS)

Capillary electrophoresis- nuclear magnetic resonance

spectroscopy(CE/NMR)

Capillary electrophoresis-surface enhanced Raman

spectrometry

(TLC-SERS)

Thin layer

chromatography(TLC)

Thin layer chromatography- mass

spectrometry(TLC/MS)

Thin layer chromatography- surface enhanced Raman

spectrometry(TLC-SERS)

Superficial fluid

chromatography/

extraction(SFC/SFE)

Superficial fluid extraction-capillary gas

chromatography-mass spectrometry(SFE-CGC-MS)

Superficial fluid-Fourier-transform infrared(SFC-FTIR)


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INTRODUCTION TO HPLC

1. Improved resolution of separated substances

2. Column packing with very small (3,5 and 10 µm) particles

3. Faster separation times (minutes)

4. Sensitivity

5. Reproducibility

6. Continuous flow detectors capable of handling small flow rates

7. Easy sample recovery, handling and maintenance.[6]

Types of HPLC Techniques

Reverse phase chromatography

Normal phase chromatography

Based on Principle of Separation

Liquid/solid chromatography (Adsorption)

Liquid/Liquid chromatography (Partition Chromatography

Ion exchange

Size exclusion

Chiral chromatography

Affinity chromatography

INSTRUMENTATION OF HPLC

Fig.1.1: Components of HPLC instrument block diagram.[22]


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Table 1.2: Physical Properties of Common Hplc Solvents.[8]

Solvent MW BP RI

(25oC)

UV

Cutoff

(nm)

Density

g/Ml

(25oC)

Viscosity

(25oC)

Dielectric

Constant

Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8

Dioxane 88.1 101 1.420 215 1.034 1.26 2.21

Ethanol 46.1 78 1.359 205 0.789 1.19 24.5

Ethylacetate 88.1 77 1.372 256 0.901 0.450 6.02

Methanol 32.0 65 1.326 205 0.792 0.584 32.7

CH2Cl2 84.9 40 1.424 233 1.326 0.44 8.93

Isopropanol 60.1 82 1.375 205 0.785 2.39 19.9

n-propanol 60.1 97 1.383 205 0.804 2.20 20.3

THF 72.1 66 1.404 210 0.889 0.51 7.58

Water 18.0 100 1.333 170 0.998 1.00 78.5

Column Selection Flow Chart.

Sample LC mode Column choice

Basic or acidic

Reverse Phase-ion pair

(allows neutral and charged

compounds to be

simultaneously analyzed)

C18, C8, C6, C4, C2, TMS, CN,

amino (not for carbonyl

compounds), phenyl, Hamilton

PRP-1 (pH 1-13).

Ionisable Ion Exchange

Anionic, cationic

Strong cations exchange,

Strong anion exchange.

Neutral

a)Normal phase

b)Reverse phase

a)Increasing the polarity of

bonded phases diol, CN, NH2,

silica alumina

b) C18, C8, phenyl, C2.

Preferred experimental conditions for the initial HPLC separation.[7]

Separation Variable Preferred Initial Choice

Column

Dimensions(length,ID)

Particle size

Stationary phase

Mobile phase

Solvents A and B

%B

Buffer (compound, PH,

concentration)

Additives (eg., amine

modifiers, ion pair reagents)

Flow rate

Temperature

Sample size

Volume

Weight

15 × 0.46 cm

5 µm a

C8 or C18

Buffer – acetonitrile

80-100 % b

25Mmpotassium phosphate,

2.0 <Ph< 3.0 c

Do not use initially

1.5 -2.0 mL/min

35-45 0C

<25 µL

<100 µg


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1. The validity of analytical method can be verified only by laboratory studies. Therefore

documentation of the successful completion of such studies is a basic requirement for

determining whether a method is suitable for its intended applications.[7]

Steps involved in development of HPLC method.[7]

Literature survey: To avoid duplication of the method and invention of new method.

Information about the solubility, the structure of the drugs and their Physico- chemical

properties for the known compounds and can be applied for the new compounds.

Selection of chromatographic method: First reversed phase should be tried. If not

successful, normal phase should be taken into consideration. For ion exchange or ion pair

chromatography, first ion suppression by pH control and reversed phase chromatography

should be tried.

Selection of stationary phase: Matching the polarity of sample and stationary phase and

using a mobile phase of different polarity achieves a successful separation.

Selection of mobile phase: Reversed phase bonded packing, when used in conjunction with

highly polar solvents. Mobile phase may be either single liquid or combination of liquids,

which are compatible with sample, column and instrument.

Selection of suitable detector: Detector is the eye of HPLC system and measures the

compounds after their separation on the column. There are basically two types of detectors:

bulk property detectors; Solute property detectors.UV detector is the first choice because of

its convenience and applicability in case of most of the samples. The latest version of

equipments is available with photo diode-array detectors.

Analytical Method Validation

Method validation as per ICH can be defined as “Establishing documented evidence, which

provides a high degree of assurance that a specific activity will consistently produce a desired

result or product meeting its predetermined specifications and quality characteristics”.


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Acceptance criteria of validation for HPLC.[22]

S.No Characteristics Acceptance criteria

1 Accuracy 98-102%

2 Precision RSD<2

3 Specificity No interference

4 Detection limit S/N >3:1

5 Quantitation limit S/N > 10:1

6 Linearity R2> 0.99

LITERATURE REVIEW

Prasanthi Chengalva, et al., (2016): Objective: To develop an accurate, precise and linear

RP-HPLC method for simultaneous quantitative estimation of Metformin hydrochloride and

Nateglinide in tablets and validate as per ICH guidelines. Methods: The method used a

reverse phase column, Inertsil C18-ODS 3V (2504.6 mm, 5 m), a mobile phase comprising of

phosphate buffer (pH 4.0): Acetonitrile: methanol (30:60:10) flow rate of 1.0 ml/min and a

detection wavelength of 221 nm using a UV detector. Results: The developed method

resulted in elution of Metformin hydrochloride at 2.45 min and Nateglinide at 4.21 min. The

calibration curves were linear (r=0.999) in the concentration range of 60-140 g/ml and 14.4-

33.2 g/ml for Metformin hydrochloride and Nateglinide respectively. The percentage

recoveries were found to be 99.59-101.36 for Metformin hydrochloride and 98.43-101.38 for

Nateglinide. The LOD was found to be 2.18 g/ml and 1.55 g/ml for Metformin hydrochloride

and Nateglinide respectively. LOQ was found to be 8.52g/ml and 4.69g/ml for Metformin

hydrochloride and Nateglinide respectively. Conclusion: A simple, accurate, precise, linear

and rapid RP-HPLC method was developed for simultaneous quantitative estimation of

Metformin hydrochloride and Nateglinide in bulk and pharmaceutical formulation and

validated as per ICH guidelines. Hence, the method holds good for the routine analysis of

Metformin hydrochloride and Nateglinide in various pharmaceutical industries as well as in

academics.


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Metformin Drug Profile[24-26]

Name Metformin

Description

This compound belongs to the class of organic compounds known as

biguanides. These are organic compounds containing two N-linked

guanidines.

Structure

Categories Anti-Diabetic Agent

Weight Average:129.1636

Chemical Formula C4H11N5

IUPAC Name 1-carbamimidamido-N,N-dimethylmethanimidamide

Classes Guanidines

Solubility

Metformin was found to be freely soluble in water; slightly soluble in alcohol;

practically insoluble in acetone and in methylene chloride.

Mechanism of

action

Metformin mechanisms of action differ from other classes of oral

antihyperglycemic agents. Metformin decreases blood glucose levels by

decreasing hepatic glucose production, decreasing intestinal absorption of

glucose, and improving insulin sensitivity by increasing peripheral glucose

uptake and utilization. These effects are mediated by the initial activation by

Metformin of AMP-activated protein kinase (AMPK), a liver enzyme that

plays an important role in insulin signaling, whole body energy balance, and

the metabolism of glucose and fats. Activation of AMPK is required for

metformin's inhibitory effect on the production of glucose by liver cells.

Increased peripheral utilization of glucose may be due to improved insulin

binding to insulin receptors. Metformin administration also increases AMPK

activity in skeletal muscle. AMPK is known to cause GLUT4 deployment to

the plasma membrane, resulting in insulin-independent glucose uptake. The

rare side effect, lactic acidosis, is thought to be caused by decreased liver

uptake of serum lactate, one of the substrates of gluconeogenesis. In those

with healthy renal function, the slight excess is simply cleared. However,

those with severe renal impairment may accumulate clinically significant

serum lactic acid levels. Other conditions that may precipitate lactic acidosis

include severe hepatic disease and acute/decompensated heart failure.

Protein binding Metformin is negligibly bound to plasma proteins.

Side Effects

Side effects of Metformin include: physical weakness (asthenia), diarrhea, gas

(flatulence), symptoms of weakness, muscle pain (myalgia), upper respiratory

tract infection, low blood sugar (hypoglycemia), abdominal pain (GI

complaints), and lactic acidosis (rare), low blood levels of vitamin B-12.

NATEGLINIDE DRUG PROFILE[27-29]

Name : Nateglinide

Description : Nateglinide is an oral antihyperglycemic agent used for the treatment of


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non-insulin-dependent diabetes mellitus (NIDDM). It belongs to the

meglitinide class of short-acting insulin secretagogues, which act by

binding to β cells of the pancreas to stimulate insulin release. Nateglinide

is an amino acid derivative that induces an early insulin response to

meals decreasing postprandial blood glucose levels.

Structure

Chemical Name : 3-phenyl-2-[(4-propan-2-yl cyclo hexane carbonyl)

amino] propanoic acid

Molecular Formula : C19H27NO3

Molecular Weight : 317.42 g/mol

Appearance : A white powder

Solubility : It is freely soluble in methanol, ethanol, and

chloroform, soluble in ether, sparingly soluble in

Acetonitrile and octanol, and practically insoluble in

water.

Melting Point : 129-130 oC

pKa : 4

Category : Hypoglycemic agent

Protein Binding : 98% bound to serum proteins, primarily serum

albumin and to a lesser extent α1 acid glycoprotein.

Metabolism : hepatic.

Half-life : 1.5 hours.

Excretion : Urine (83%) and feces (10%).


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Mechanism of Action

Nateglinide activity is dependent on the presence functioning β cells and glucose. In contrast

to sulfonylurea insulin secretatogogues, nateglinide has no effect on insulin release in the

absence of glucose. Rather, it potentiates the effect of extracellular glucose on ATP-sensitive

potassium channel and has little effect on insulin levels between meals and overnight. As

such, nateglinide is more effective at reducing postprandial blood glucose levels than fasting

blood glucose levels and requires a longer duration of therapy (approximately one month)

before decreases in fasting blood glucose are observed. The insulinotropic effects of

nateglinide are highest at intermediate glucose levels (3 to 10 mmol/L) and it does not

increase insulin release already stimulated by high glucose concentrations (greater than

15mmol/L). Nateglinide appears to be selective for pancreatic β cells and does not appear to

affect skeletal or cardiac muscle or thyroid tissue.

Side Effects

An overdose may result in an exaggerated glucose-lowering effect with the development of

hypoglycemic symptoms.

Food Interactions: Take up to 30 minutes before meals.

DRUG FORMULATION

S.No. Drug name Label Claim Brand name Company

1 Nateglinide 120 mg Starlix Novartis

Combined Drug Formulation

S.No. Drug name Label Claim Brand Name Company

1 Metformin/Nateglinide 60mg/500mg Trunate Plus Aar Ess Remedies

Private Limited

AIM AND OBJECTIVE

Existing literature reveals that Metformin and Nateglinide can be analyzed by HPLC using

UV detection, TLC, HPTLC and HPLC in bulk and combined tablet dosage forms.

A comprehensive, validated and simple analytical method development and validation of

Metformin and Nateglinide in bulk and its tablet dosage forms, therefore, crucial. HPLC with

PDA detector is a good selection as PDA detector is available in most laboratories.


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Therefore, in proposed project a successful attempt has been made to develop, simple,

accurate, and economic methods for analysis of Metformin and Nateglinide in bulk and

combined tablet dosage forms was developed and validated as per the ICH Guidelines.

OBJECTIVE

The objective of the present work is to development and validates a RP-HPLC method with

PDA detector for the development and validation Metformin and Nateglinide in bulk and

combined tablet dosage forms.

In the method development of Metformin and Nateglinide we have decided to carry out our

project work by incorporating the reverse phase high performance liquid chromatography

(RP-HPLC).

Then the developed method will be validated according to ICH guidelines for its various

parameters.

PLAN OF WORK

In order to develop a simple, reliable and an accurate method development and validation of

Metformin and Nateglinide in bulk and pharmaceutical dosage form by reverse phase HPLC

and validate the method for its repeatability and reproducibility

Plan of the proposed work includes the following steps

Selection of drug and literature survey.

Solubility studies and optimization of conditions.

Analytical method(s) development using HPLC etc.

Assay of the drugs(s) in marketed formulations using the proposed method(s).

Procurement of raw materials.

Establishment of system suitability parameters.

Trails for the method development of Metformin and Nateglinide.

Setting of the optimized method.

Validation of the optimized method for Metformin and Nateglinide.

Validation parameters include:

System suitability

Specificity

Method precision


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Linearity

Accuracy

Range

Robustness

EXPERIMENTAL METHODS

Instruments Used

Table: Instruments used.

S.No. Instruments And Glass wares Model

1 HPLC WATERS Alliance 2695 separation module,

Software: Empower 2, 996 PDA detector.

2 pH meter Lab India

3 Weighing machine Sartorius

4 Volumetric flasks Borosil

5 Pipettes and Burettes Borosil

6 Beakers Borosil

7 Digital ultra sonicator Labman

CHEMICALS USED

Table: Chemicals used.

S.No Chemical Brand names

1 Metformin (Pure) Sura labs

2 Nateglinide (Pure) Sura labs

3 Water and Methanol for HPLC LICHROSOLV (MERCK)

4 Acetonitrile for HPLC Merck

HPLC METHOD DEVELOPMENT

TRAILS

Preparation of standard solution

Accurately weigh and transfer 10 mg of Metformin and Nateglinide working standard into a

10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and

removal of air completely and make volume up to the mark with the same Methanol.

Further pipette 0.6ml of Metformin and 1ml of Nateglinide from the above stock solutions

into a 10ml volumetric flask and dilute up to the mark with Methanol.

Optimized Chromatographic Conditions

Instrument used : Waters Alliance 2695 HPLC with PDA Detector 996 model.

Temperature : 38ºC

Column : Symmetry ODS C18 (4.6mm×150mm) 5µm Particle Size


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Mobile phase : Methanol: 0.1% Orthophosphoric acid (64:36% v/v)

Flow rate : 1ml/min

Wavelength : 224nm

Injection volume : 20µl

Run time : 7.0minutes

METHOD VALIDATION

PREPARATION OF MOBILE PHASE

Preparation of mobile phase

Accurately measured 640ml of Acetonitrile (64%) of and 360ml of HPLC Water (36%) were

mixed and degassed in a digital ultrasonicater for 15 minutes and then filtered through 0.45 µ

filter under vacuum filtration.

Diluent Preparation

The Mobile phase was used as the diluent.

VALIDATION PARAMETERS

System Suitability


10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it

completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette out 0.6ml of Metformin and 1ml of Nateglinide from the above stock

solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

SPECIFICITY STUDY OF DRUG

Preparation of Standard Solution


10ml of clean dry volumetric flasks add about 7ml of Diluents and sonicate to dissolve it




Preparation of Sample Solution

Take average weight of Tablet and crush in a mortar by using pestle and weight 10 mg

equivalent weight of Metformin and Nateglinide sample into a 10mL clean dry volumetric


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flask and add about 7mL of Diluent and sonicate to dissolve it completely and make volume

up to the mark with the same solvent. Filter the sample solution by using injection filter

which contains 0.45µ pore size.



%ASSAY =

Sample area Weight of standard Dilution of sample Purity Weight of tablet

___________ × ________________ × _______________×_______×______________×100

Standard area Dilution of standard Weight of sample 100 Label claim

Preparation of Drug Solutions For Linearity




Preparation of Level – I (20ppm of Metformin and 60ppm of Nateglinide)

Pipette out 0.2ml of Metformin and 0.6ml of Nateglinide in to a 10ml volumetric flask and

make the volume upto mark by using diluent and sonicate for air entrapment.

Preparation of Level – II (40ppm of Metformin and 80ppm of Nateglinide)

Pipette out 0.4ml of Metformin and 0.8ml of Nateglinide in to a 10ml volumetric flask and


Preparation of Level – III (60ppm of Metformin and 100ppm of Nateglinide)

Pipette out 0.6ml of Metformin and 1ml of Nateglinide in to a 10ml volumetric flask and


PRECISION

Repeatability

Preparation of Metformin and Nateglinide Product Solution for Precision







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The standard solution was injected for five times and measured the area for all five injections in

HPLC. The %RSD for the area of five replicate injections was found to be within the specified

limits.

Intermediate Precision

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision

was performed on different days by maintaining same conditions.

Procedure

DAY 1

The standard solution was injected for Six times and measured the area for all Six injections in

HPLC. The %RSD for the area of Six replicate injections was found to be within the specified

limits.

DAY 2

The standard solution was injected for Six times and measured the area for all Six injections in

HPLC. The %RSD for the area of Six replicate injections was found to be within the specified

limits.

ACCURACY

For preparation of 50% Standard stock solution

Accurately weigh and transfer 10mg of Metformin and Nateglinide working standard into a



Further pipette out 0.3ml of Metformin and 0.5ml of Nateglinide from the above stock


For preparation of 100% Standard stock solution







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ROBUSTNESS

The analysis was performed in different conditions to find the variability of test results. The

following conditions are checked for variation of results. .

For preparation of Standard solution






Effect of Variation of flow conditions

The sample was analyzed at 0.9 ml/min and 1.1 ml/min instead of 1ml/min, remaining

conditions are same. 20µl of the above sample was injected and chromatograms were

recorded.

Effect of Variation of mobile phase organic composition

The sample was analyzed by variation of mobile phase i.e. Methanol: 0.1% Orthophosphoric

acid (64:36% v/v) was taken in the ratio and 69:31, 59:41 instead of 64:36 remaining

conditions are same. 20µl of the above sample was injected and chromatograms were

recorded.

RESULTS AND DISCUSSION

Trial 1: (Combination drug trails:-Metformin+ Nateglinide).

Mobile phase : Methanol + Water (40:60%v/v)

Column : Thermo BDS Hypersil C18 column having 250 x 4.6mm 5μ,

Flow rate : 0.5ml/min

Wavelength : 224 nm

Column temp : Ambient

Injection Volume : 10 µl

Run time : 6minutes


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Figure-: Chromatogram for Trail 1.

Table: Peak Results for Trail 1.

S.No Peak Name Rt Area Height USP

Resolution

USP

Tailing

USP Plate

count

1 Metformin 2.476 648745 58794 1.24 1254

Trail 2.

Mobile phase : Acetonitrile: Methanol (40% -60%v/v)

Column : Zorbax C18 (4.6mm×250mm) 5µmparticle size

Flow rate : 0.8 ml/min

Wavelength : 224 nm


Sample Temp : Ambient


Run time : 9 minutes

Figure: Chromatogram for Trail 2.


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S.

No. Peak Name Rt Area

Heig

ht

USP

Resolution

USP

Tailing

USP plate

count

1 Metformin 2.485 536521 59898 1.13 4265

2 Nateglinide 4.030 8658452 87984 2.35 1.26 3412

Trail 3

Mobile phase : Acetonitrile: 0.1% Orthophosphoric acid (70:30% v/v)

Column : Develosil C18 (4.6mm×250mm, 5µm)


Wavelength : 224 nm



Injection Volume : 0.9µl/min




S.

No Peak Name Rt Area

Heig

ht

USP

Resolution

USP

Tailing

USP plate

count

1 Nateglinide 1.573 1852456 52365 1.26 2653

2 Metformin 4.611 125875 2546 4.35 0.92 4536

Trail 4





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Wavelength : 224 nm

Column temp : 360C


Injection Volume : 1.0µl/min

Run time : 10 minutes.



S. No Peak Name Rt Area Height USP

Resolution

USP

Tailing

USP plate

count

1 Nateglinide 6.025 4265985 325685 0.96 1365.6

2 Metformin 7.977 53625 45685 2.51 1.08 3856.5

Trial 5: (Optimized Condition)



Flow rate : 1 ml/min

Wavelength : 224 nm

Column temp : 38ºC





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Figure: Chromatogram for Trail 5.


S.

No Peak name Rt Area Height

USP

Resolution

USP

Tailing

USP plate

count

1 Metformin 2.808 65258 4326 1.08 5685.4

2 Nateglinide 3.880 8659854 659823 5.68 1.42 6895.7

METHOD VALIDATION PARAMETERS

Assay (Standard)

Fig: Chromatogram showing assay of standard injection-1.


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Assay (Sample)

Fig: Chromatogram showing assay of sample injection -1.

Precision

Precision of the method was carried out for both sample and standard solutions as described

under experimental work. The corresponding chromatograms and results are shown below.

Fig: chromatogram for standard injection -1.

Intermediate Precision/Ruggedness

There was no significant change in assay content and system suitability parameters at

different conditions of ruggedness like day to day and system to system variation.


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Fig-: chromatogram for sample injectiocn-1.

Acceptance Criteria

%RSD of five different sample solutions should not more than 2.

The %RSD obtained is within the limit, hence the method is rugged.

ACCURACY

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the

% recovery was calculated.

Accuracy Standard

Figure: Chromatogram for Accuracy std. Injection-1.


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LINEARITY

The linearity range was found to lie from 20-100ppm of Metformin, 60µg/ml to 140µg/ml of

Nateglinide and chromatograms are shown below.

Figure 6.3.4 calibration graph for Metformin.

QUANTITATION LIMIT

The quantitation limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined.

LOQ=10×σ/S

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

Metformin

Result

=2.91µg/ml

Nateglinide

Result

= 6.18µg/ml

ROBUSTNESS

The standard and samples of Metformin and Nateglinide were injected by changing the

conditions of chromatography. There was no significant change in the parameters like

resolution, tailing factor, asymmetric factor, and plate count.


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Variation in flow

Figure: Chromatogram showing less flow of 0.9ml/min.

Table: System Suitability Results for Metformin.

S.No Change in Organic Composition

in the Mobile Phase

System Suitability Results

USP Plate Count USP Tailing

1 10% less 5895.3 1.12

2 *Actual 5685.4 1.08

3 10% more 5964.2 1.16

Table: System suitability results for Nateglinide.

S.No Change in Organic Composition

in the Mobile Phase

System Suitability Results

USP Plate Count USP Tailing

1 10% less 6785.2 1.46

2 *Actual 6895.7

1.42,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

,,,,,,,,,,,,,,,,,,,,,,,,,,,,

3 10% more 6982.4 1.49,

CONCLUSION

The study is focused to develop and validate HPLC methods for estimation of Metformin and

Nateglinide in bulk and tablet dosage form.

For routine analytical purpose it is desirable to establish methods capable of analyzing huge

number of samples in a short time period with good robustness, accuracy and precision

without any prior separation steps. HPLC method generates large amount of quality data,

which serve as highly powerful and convenient analytical tool.


903


The method shows good reproducibility and good recovery. From the specificity studies, it

was found that the developed methods were specific for Metformin and Nateglinide.

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