Chapter-5 Simultaneous estimation of vildagliptin and ...shodhganga.inflibnet.ac.in/bitstream/10603/20011/11... · metformin treatment fails to achieve glycemic goals [33]. When used

Chapter-5

Simultaneous

estimation of

vildagliptin and

metformin by

RP – HPLC

5.1 Introduction

Vildagliptin is a drug for the treatment of diabetes, whose chemical name is (S)

[N-(3-hydroxy-1-adamantyl) glycyl] pyrrolidine

vildagliptin is C17H25N3O2, its molecular weight is 303.399 g mol

hyperglycemic agent (anti-diabetic drug) of the new dipeptidyl peptidase

inhibitor class of drugs.Vildagliptin inhibits the inactivation of g

[1]and glucose-dependent insulinotropic pep

to potentiate the secretion of insulin in the beta cells and suppress glucagon release by the alpha

cells of the islets of langerhans in the pancreas. Vildagliptin has been shown to reduce

hyperglycemia in type 2 diabetes mellitus. It is used to treat type 2 or non

diabetes mellitus (NIDDM). The chemical structure of vildagliptin is as shown in Figure 5.1.

Figure 5.1: Structure of vildagliptin

Vildagliptin works by increasing the amount

called glucagon-like peptide-1 (GLP

These hormones are normally produced naturally by the body in response to food intake. Their

function is to control blood sugar (glucose) levels.GLP

help to control blood glucose.Firstly, they stimulate the pancreas to produce insulin in response

to increasing levels of glucose in the blood. (Insulin is the main hormone responsible for

controlling sugar levels in the blood. It causes cells in the body to remove sugar from the blood).

GLP-1 also reduces the production of glucagon. (Glucagon is a hormone that normally increases

glucose production by the liver). GLP

body called dipeptidyl peptidase

preventing it from breaking down the GLP

hormones in the body and so increases their ef

Vildagliptin is a drug for the treatment of diabetes, whose chemical name is (S)

adamantyl) glycyl] pyrrolidine-2-carbonitrile. The empirical formula of

its molecular weight is 303.399 g mol-1. Vildagliptin

diabetic drug) of the new dipeptidyl peptidase

class of drugs.Vildagliptin inhibits the inactivation of glucagon like peptide

dependent insulinotropic peptide (GIP)[2] by DPP- IV, allowing GLP



ype 2 diabetes mellitus. It is used to treat type 2 or non-insulin dependent


Figure 5.1: Structure of vildagliptin

Vildagliptin works by increasing the amount of two incretin hormones found in the body,

1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP).


d sugar (glucose) levels.GLP-1 and GIP have two main actions that

Firstly, they stimulate the pancreas to produce insulin in response


ontrolling sugar levels in the blood. It causes cells in the body to remove sugar from the blood).

1 also reduces the production of glucagon. (Glucagon is a hormone that normally increases

glucose production by the liver). GLP-1 and GIP are normally broken down by an enzyme in the

body called dipeptidyl peptidase-4 (DPP-4). Vildagliptin works by binding to this enzyme and

preventing it from breaking down the GLP-1 and GIP. This increases the levels of these

hormones in the body and so increases their effect on controlling blood sugar.

Vildagliptin is a drug for the treatment of diabetes, whose chemical name is (S)-1-

empirical formula of

Vildagliptin is an oral anti-

diabetic drug) of the new dipeptidyl peptidase-IV (DPP-IV)

lucagon like peptide-1 (GLP-1)

, allowing GLP-1 and GIP



insulin dependent


of two incretin hormones found in the body,

dependent insulinotropic peptide (GIP).


1 and GIP have two main actions that

Firstly, they stimulate the pancreas to produce insulin in response


ontrolling sugar levels in the blood. It causes cells in the body to remove sugar from the blood).

1 also reduces the production of glucagon. (Glucagon is a hormone that normally increases

oken down by an enzyme in the

4). Vildagliptin works by binding to this enzyme and

1 and GIP. This increases the levels of these

Diabetes affects the control of blood sugar levels. In diabetes, the body may not be able

to produce enough insulin or the insulin that it produces may not have the full effect. In some

instances, the body may not be able to produce any insulin at all. Insulin helps the body to use

the sugar in the blood properly and it helps to prevent blood sugar levels from becoming too

high. Glucagon is another hormone which is important in controlling blood sugar levels.

Glucagon causes blood sugar levels to rise when they are low. Vildagliptin increases the amount

of insulin produced by the body. It also decreases the amount of glucagon which is produced by

the body. Because of these effects, vildagliptin can help to control blood sugar levels in people

with diabetes. Vildagliptin is used in combination with other medicines which help to control

blood sugar levels. Dipeptidyl peptidase-4 inhibitors have a low incidence of hypoglycemia

without significant weight gain and there is strong evidence that the administration of

vildagliptin results in improved α- and β-cell function. Data suggest that DPP-4 inhibitors might

also have a role in the setting of myocardial infarction (heart attack) and lipid management, and

in the prevention of type 2 diabetes [3]. Good tolerability and clinically relevant improvements

in glycemic control have also been observed with vildagliptin as an add-on treatment to

sulfonylurea, thiazolidinedione, or insulin treatment [4]. The combination of metformin and

vildagliptin offers advantages when compared to currently used combinations with additive

efficacy and complimentary mechanisms of action, since it does not increase the risk of

hypoglycemia and does not promote weight gain [5]. Sustained efficacy and reduced

hypoglycemia during one year of treatment with vildagliptin added to insulin in patients with

type 2 diabetes mellitus. [6]. As reviewed herein, clinical trials have shown that vildagliptin

improves glycemic control in patients with type 2 diabetes mellitus as monotherapy [7 - 11] and

as add-on or initial combination therapy with other oral antidiabetic agents and insulin [12, 13].

Vildagliptin improves the sensitivity of the alpha cell to glucose in patients with type 2 diabetes

mellitus (T2DM) by enhancing the alpha-cell responsiveness to both suppressive effects of

hyperglycaemia and stimulatory effects of hypoglycaemia. Numerous long-term clinical trials of

up to two years have demonstrated that vildagliptin 50 mg once daily or twice daily is effective,

safe and well tolerated in patients with T2DM as either monotherapy or in combination with a

variety of other anti-diabetic agents [14].

Metformin hydrochloride, chemically known as N, N-dimethylimidodicarbonimidic

diamide hydrochloride, is an orally administered biguanide, widely used in the treatment of type

2 (non-insulin dependent) diabetes mellitus.

been conclusively shown to prevent

reduce low density lipoprotein (LDL) cholesterol

with weight gain. It is also used in the treatment of

investigated for other diseases where

works by suppressing glucose production by the liver.

widely prescribed antidiabetic drug in the world, in the United States alo

prescriptions were filled in 2010 for its

early treatment with metformin is associated with reduced cardiovascular morbidity and total

mortality in newly diagnosed type 2 diabe

2 diabetes, but is increasingly being used in

alcoholic fatty liver disease (NAFLD) [18]

risk of hypoglycemia than the sulfonylureas [20] and also not associated with weight gain, and

modestly reduces LDL and triglyceride

The nucleophilicity of metformin hydrochloride is moderate and that is a desired

property. Metformin is shown to belong to a new class of compounds called nitreones.

empirical formula is C4H11N5, molecular weight is 129.16

shown in Figure 5.2.

Metformin is a first-line treatment option because it has positive effects not only on

anovulation, but also on insulin resistance, hirsutism, and obesity often associated

with polycystic ovary syndrome (PCOS) [22]. Several

controlled trials have found that metformin is as effective and safe as insulin for the management

of gestational diabetes [23 -

insulin dependent) diabetes mellitus. Metformin is the only anti diabetic drug that has

been conclusively shown to prevent the cardiovascular complications of diabetes. It helps to

(LDL) cholesterol and triglyceride levels, and is not associated

with weight gain. It is also used in the treatment of polycystic ovary syndrome,

ated for other diseases where insulin resistance may be an important factor.Metformin

works by suppressing glucose production by the liver. Metformin is now believed to be the most

widely prescribed antidiabetic drug in the world, in the United States alone, more than 48 million

prescriptions were filled in 2010 for its generic formulations [15]. There is some evidence that


mortality in newly diagnosed type 2 diabetic patients [16]. Metformin is primarily used for type

2 diabetes, but is increasingly being used in polycystic ovary syndrome (PCOS) [17],

(NAFLD) [18] and premature puberty [19]. Metformin has a lower

ia than the sulfonylureas [20] and also not associated with weight gain, and

triglyceride levels [21].


property. Metformin is shown to belong to a new class of compounds called nitreones.

, molecular weight is 129.16 g mol-1 and its chemical stru

Figure 5.2: Structure of metformin

line treatment option because it has positive effects not only on


polycystic ovary syndrome (PCOS) [22]. Several observational studies and randomized,


25]. Observational studies conducted by th

Metformin is the only anti diabetic drug that has

the cardiovascular complications of diabetes. It helps to

levels, and is not associated

polycystic ovary syndrome, and has been

may be an important factor.Metformin

Metformin is now believed to be the most

ne, more than 48 million

. There is some evidence that


tic patients [16]. Metformin is primarily used for type

(PCOS) [17], non-

Metformin has a lower

ia than the sulfonylureas [20] and also not associated with weight gain, and


property. Metformin is shown to belong to a new class of compounds called nitreones. Its

and its chemical structure is as

line treatment option because it has positive effects not only on


and randomized,


25]. Observational studies conducted by the University of

Dundee have shown a decrease of 25–37% in cancer cases in diabetics taking metformin [26,

27]. Metformin activates adenosine monophosphate activated protein kinase (AMPK), an

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

metabolism of glucose and fats [28]. Activation of AMPK is required for metformin's inhibitory

effect on the production of glucose by liver cells [29]. Metformin has acid dissociation constant

values (pKa) of 2.8 and 11.5 and, therefore, exists very largely as the hydrophilic cationic

species at physiological pH values. The acid dissociation constant values make metformin a

stronger base than most other basic drugs with less than 0.01% unionized in blood. Metformin

absorption is the rate-limiting step in drug disposition because absorption is transporter

dependent and saturatable, which causes bioavailability to diminish as dosage increases [30].

Metformin has an oral bioavailability of 50–60% under fasting conditions, and has a limited

window for absorption. Its absorption is incomplete, prolonging the intestinal transit time

increases absorption [31]. Metformin is not metabolized and it is cleared from the body

by tubular secretion and excreted unchanged through urine, metformin is undetectable in blood

plasma within 24 hours of a single oral dose [32].

Current guidelines encourage a prompt move to combination treatment when initial

metformin treatment fails to achieve glycemic goals [33]. When used for type 2 diabetes,

metformin is often prescribed in combination with other drugs. Several are available as fixed-

dose combinations, also with the purpose of reducing pill burden and making administration

simpler and more convenient [34].The vildagliptin-metformin association seems to have

favorable effects on beta-cell function and is characterized by good safety and tolerability

profiles when compared with other antidiabetic agents. The available data suggest that

administration of fixed-dose combination products, together with the low incidence of adverse

gastrointestinal events, may improve compliance and adherence of patients to therapy, resulting

in an improved metabolic control [35]. In treatment-naive patients, combinations of vildagliptin

and both high-dose and low-dose metformin provide superior efficacy to monotherapy

treatments with a comparable overall tolerability profile and low risk of hypoglycaemia [36].

Vildagliptin add-on to metformin has similar efficacy and reduced hypoglycaemic risk compared

with glimepiride, with no weight gain [37]. The addition of vildagliptin to metformin gave a

better improvement of glycemic control, insulin resistance, and β-cell function compared with

metformin alone [38]. The complementary mechanisms of action of the two agents in

combination have been shown to provide additive and sustained reductions in hemoglobin

A1c compared with metformin monotherapy. In active-controlled trials, the vildagliptin-

metformin combination has been shown to produce equivalent reductions in hemoglobin A1c to

pioglitazone-metformin and glimepiride-metformin combinations, without significant risk of

hypoglycemia and without causing weight gain [39, 40]. Vildagliptin offers a clinically

important outcome when added to metformin with a twice daily dose regimen, taking advantage

of its tight binding and slow dissociation characteristics that lead to a sustained overnight effect

[41]. In patients with type2 diabetes mellitus (T2DM), inadequately controlled with metformin

up to 1000 mg daily, the addition of vildagliptin, 100 mg daily, achieved larger hemoglobin

A1c reduction with fewer gastrointestina events than with increasing the metformin dose [42].

Literature survey reveals that a number of methods were reported for the determination of

vildagliptin and metformin separately. Bagary et al reported spectrophotometric methods for the

determination of sitagliptin and vildagliptin in bulk and dosage forms [43] and also reported a

liquid chromatographic method for the determination of vildagliptin [44]. A chromatographic

method was reported by Pharne et al for the determination of vildagliptin by using reverse

phased high performance liquid chromatography (RP-HPLC) [45]. Mousumi Kar et al reported a

HPLC method for the estimation of metformin hydrochloride in formulated dosage forms [46]. A

method for the determination of metformin hydrochloride in human plasma and urine by using a

HPLC with conventional octadecylsilane column was reported by Raniah et al [47]. Narendra

kumar et al reported a HPLC method for the determination of metformin hydrochloride in

pharmaceutical dosage forms [48]. Valentina Porta et al reported a HPLC-UV method for the

determination of metformin in human plasma [49]. Very few analytical methods were reported

for simultaneous determination of vildagliptin and metformin in pharmaceutical formulations.

Pushpalatha et al reported a method for simultaneous estimation of vildagliptin and metformin in

tablet dosage forms by using reverse phased HPLC in which the retention times were achieved

for vildagliptin at about 2.1 minutes and metformin at about 5.6 minutes with chromatographic

run time of 8 minutes [50]. Mohammad Abdul et al developed a reversed phase liquid

chromatographic method for simultaneous determination of two gliptins in their binary mixtures

with metformin [51]. Usha rani et al reported a method for simultaneous determination of

vildagliptin and metformin in bulk and pharmaceutical formulations by using UV

spectrophotometer [52]. RP-HPLC method developed for the determination of metformin and

vildagliptin in bulk, pharmaceutical formulation and bio analytical studies by Alekya et al [53] in

which the retention times of metformin and vildagliptin are about 2.6 and 4.3 respectively and

the response of vildagliptin peak was found very low with asymmetric peak shape. Shelke et al

reported a method for simultaneous determination of metformin HCl and vildagliptin in

pharmaceutical formulation by using RP-HPLC in which the peak shape of metformin and

vildagliptin were found asymmetric [54]. Ishaq et al reported a method for simultaneous

determination of metformin and vildagliptin by using RP-HPLC in which the runtime is about 7

minutes [55].

The literature survey revealed that methods were reported for the individual

determination of vildagliptin and metformin in pharmaceutical formulations. A few methods

were reported for the estimation of vildagliptin combined with other molecules in a combined

formulation [56]. Less number of methods was reported for the simultaneous determination of

metformin and other in a combined pharmaceutical dosage units [57, 58, 59]. The method

reported by Mohammed Abdul et al [51] for the simultaneous determination of vildagliptin and

metformin in a combined pharmaceutical formulation is found to be more suitable and

appropriate when compared with other reported methods. The analysis of vildagliptin and

metformin in pharmaceutical drug substances and formulations was carried out by using

chromatographic technique in the reported method. Although reported method is quite suitable

for the simultaneous determination of vildagliptin and metformin in pharmaceutical

formulations, the run time of chromatographic analysis is found as 12.5minutes. Therefore, it is

very imperative to develop a suitable analytical method for simultaneous determination of

vildgliptin and metformin such that the methods could be easily adapted for routine and in-

process quality control analysis or similar studies with less analysis time.

The aim of this study is to develop a rapid, simple, precise and accurate reverse phase

high performance liquid chromatographic (RP-HPLC) method for the simultaneous

determination of vildagliptin and metformin in pharmaceutical formulations. The developed

method is validated as per the regulatory requirement to use for routine quality control

applications. The proposed method is validated according to ICH guidelines [60] in terms of

specificity, precision, accuracy, linearity, and robustness.

5.2. Experimental

5.2.1. Reference substances, chemicals, reagents and samples

Pharmaceutical grade vildagliptin, metformin active pharmaceutical ingredient (API) and

combined formulated tablets were procured from Novartis limited, Hyderabad, Andhra Pradesh,

India. Analytical grade potassium dihydrogen phosphate and ortho phosphoric acid were

purchased from Fischer scientific chemicals, Mumbai, HPLC grade acetonitrile and methanol

were procured form Ranbaxy Pvt. limited, Delhi. 0.45µm membrane filter, purchased from

millipore, Barcelona was employed in the study. The entire experiment was performed using

“class A” volumetric glassware and HPLC grade water.

5.2.2. Instrumentation

Simultaneous determination of vildagliptin and metformin was performed by using

Waters HPLC (Milford, MA, USA) PDA system consisting of a quaternary solvent manager, a

sample manager, column-heating compartment, and photodiode array detector. This system was

controlled and output signal was monitored by Waters empower software. Thermo Hypersil

ODS, 250mm length, 4.6mm internal diameter column with particle size of 5µm porous silica

was employed as stationary phase for chromatographic separation. Sartorius semi micro balance

was used for all weighing and Labindia pH meter was used for buffer pH adjustment and

sonication was carried out by using Fastclean ultrasonic bath. All samples were filtered through

0.45 µm membrane millipore filters.

5.2.3. Blank, standard and sample solution preparation

Vildagliptin, metformin API, tablets and the corresponding placebo (without API)

were used throughout the development and validation. All the samples were treated according to

test solution preparation.

5.2.3.1. Standard solution preparation

The standard solution of vildagliptin and metformin was prepared by dissolving an

accurately weighed 500mg of vildagliptin working standard and 5mg of metformin working

standard in 50 ml of mobile phase in a 100ml volumetric flask. The contents were sonicated for

15 to dissolve the material and made up to the mark with mobile phase. The prepared standard

solution was filtered through 0.45 µm filter paper.

5.2.3.2.Blank solution preparation

Mobile phase was used as blank as it is being used as diluent for standard and sample

preparations.

5.2.3.3. Sample solution preparation

The formulated sample solution was prepared by powdering 10 tablets, weighing the

resultant powder accurately equivalent to single dosage unit and transferring into 100ml

volumetric flask. The powder was dissolved in 50ml of mobile phase, sonicated for about 15

minutes and made up to the mark with mobile phase. The prepared sample solution was filtered

through 0.45 µm filter paper. The bulk sample solution was prepared as mentioned in the

standard solution preparation (5.2.3.1) by replacing the working standard with sample material.

5.2.4. Chromatographic conditions

The analysis was carried out by using high performance liquid chromatography (HPLC).

The vildagliptin and metformin were separated on Thermo Hypersil ODS column, 250mm

length, 4.6mm internal diameter packed with porous silica of particle size 5µm, at 30°C column

oven temperature. The flow rate was maintained at 1.0 mL minute−1. The separation was

achieved by isocratic elution with a run time of 5 minutes. The mobile phase was filtered through

a 0.45µm Millipore filter, before use. UV detection was performed at 263nm. The sample

injection volume was 10 µL. The degassed composition of potassium dihydrogen phosphate

(transferred 17.418 gm of potassium dihydrogen phosphate into a beaker dissolve and diluted up

to 1000 ml volume with water and adjusted pH to 7.0 with ortho phosphoric acid) and

acetonitrile in 600:400 v/v ratio was used as the mobile phase.

5.2.5. Evaluation of blank

10µL of blank solution (mobile phase) were injected into high performance liquid

chromatograph and the chromatogram was recorded.

5.2.6. Evaluation of system suitability

10µL of standard preparation was injected into chromatographic system for five times

and evaluated the relative standard deviation of vildagliptin and metformin area responses

individually. The tailing factor, theoretical plate count and relative standard deviation were also

evaluated for both vildagliptin and metformin analyte peaks.

5.2.7. Procedure

The standard preparation and the sample preparation were separately injected into the

high performance liquid chromatograph and the area of major peaks was recorded. The diluent

chromatogram was examined for any extraneous peaks and the corresponding peaks observed in

the sample chromatogram were ignored. The retention time of vildagliptin and metformin peaks

under the present chromatographic conditions was about 2.0 and 3.5 minutes respectively.

5.2.8. Quantitation

Vildagliptin and metformin peak areas were recorded for standard and sample injections.

Respective peak areas were taken into account to quantitate the amount of vildagliptin and

metformin present in the sample as follows:

AVt CVs PV % of vildagliptin = ------ x ------- x ------ x 100 AVs CVt 100 Where, AVt=Vildagliptin peak area obtained from the sample preparation;

AVs = Vildagliptin peak area obtained from the standard preparation;

CVt = Concentration of vildagliptin in standard solution;

CVs = Concentration of vildagliptin in sample solution and

PV = Vildagliptin working standard purity in percentage.

AMt CMs PM % of metformin = ------ x ------- x ------ x 100 AMs CMt 100 Where, AMt = Metformin peak area obtained from the sample preparation;

AMs = Metformin peak area obtained from the standard preparation;

CMt = Concentration of metformin in standard solution;

CMs = Concentration of metformin in sample solution and

PM = Metformin working standard purity in percentage.

5.3. Results and discussion:

5.3.1. Method development and optimization

The objective of this work is to develop a rapid, simple and precise method for the

simultaneous determination of vildagliptin and metformin (Active Pharmaceutical Ingredient)

present in combined formulation (drug product) of vildagliptin and metformin by using a high

performance liquid chromatograph (HPLC). The method development was initiated by the

review of literature survey and studies on physical and chemical characteristics of vildagliptin

and metformin. Vildagliptin is a white to slightly yellowish crystalline power with a melting

point of approximately 150°C and it is freely soluble in water. Metformin hydrochloride is a

white to off white powder with a melting point of approximately 224°C and also freely soluble

in water. Phosphate buffer was found suitable to use as mobile phase to cope up for the injection

load on the column. Acetonitrile was found suitable as an organic modifier as it is a weak

hydrogen acceptor. The participation of residual silanol groups in the retention process is more

pronounced in acetonitrile. Based on spectral profile and absorption characteristics of

vildagliptin and metformin, UV detector with 263 nm wavelength was selected to detect both

vildagliptin and metformin. The chromatographic conditions were optimized by performing the

following trial experiments.

5.3.1.1. Optimization of chromatographic parameters:

� Trial – 1

The analysis carried out by using high performance liquid chromatography (HPLC) with

the following chromatographic conditions.

Column: Thermo Hypersil ODS column, 250mm length, 4.6mm internal diameter and

porous silica packed with particle size of 5µm.

Buffer: 17.418 gm of potassium dihydrogen phosphate were dissolved in 1000ml water

and adjusted the pH to 7.0 with ortho phosphoric acid.

Mobile phase: The degassed composition of potassium dihydrogen phosphate and

acetonitrile in 90:10 v/v respectively, filtered through 0.45 µm membrane filter.

Flow rate: 1.0 mL minute-1

Injection volume: 20 µL

Data acquisition time: 25 minutes

Detection mode:PDA 200 to 400 nm.

In this trial, metformin and vildagliptin were separated. The tailing factor obtained from

the analyte peaks was more and absorbance maxima observed at 263 nm. The elution time of

metformin and vildagliptin were found to be 11 and 16 minutes respectively.

� Trial - 2

To reduce the tailing factor and to improve the peak shape of analyte peaks, the

composition of buffer and acetonitrile was modified and wave length fixed as 263 nm for

detection.



Buffer: 17.418 gm of potassium dihydrogen phosphate were dissolved in 1000ml of water

and the pH of the solution was adjusted to 7.0 with ortho phosphoric acid.

Mobile phase: The degassed potassium dihydrogen phosphate solution and acetonitrile

were mixed in the ratio 80:20 v/v respectively and filtered through 0.45 µm membrane

filter.


Injection Volume: 20 µL


Detection mode: Ultra violet detection at 263 nm.

In this trial, the metformin and vildagliptin peak tailing was found moderate and peak

response was found to be on higher side. The retention time of metformin and vildagliptin were

observed at 5 minutes and 8 minutes respectively.

� Trial - 3

To avoid high response with symmetric peak shape, the injection volume was reduced to

10µL and also organic modifier concentration was increased in mobile phase to reduce the

retention time of analyte peaks to achieve shorter run time of chromatographic analysis.



Buffer: Potassium dihydrogen phosphate (17.418 gm in 1000mL of water) adjusted to pH

7.0 with otho phosphoric acid.

Mobile phase: Potassium dihydrogen phosphate and acetonitrile in 60:40 v/v ratio.


Injection volume: 10 µL


Detection mode: Ultra violet detection at 263 nm.

In this trial, the tailing factor and theoretical plate count of analyte peaks were found to

be satisfactory and a symmetric peak was obtained. The retention times of metformin and

vildagliptin were found to be at about 2.1 and 3.5minutes respectively with suitable peak

response.

Based on the results of all the trails made above, the results obtained under the optimal

conditions of trial 3 were found to be satisfactory for simultaneous assay determination of

metformin and vildagliptin. Hence, it was finally concluded that the best optimal conditions for

the simultaneous quantitative separation of metformin and vildagliptin are Thermo Hypersil

ODS column of 250mm length, 4.6mm internal diameter and packed with particle size of 5µm

porous silica, at 30°C column oven temperature with a flow rate of 1.0 mL minute−1. The

separation was achieved by isocratic elution with run time of 5 minutes. The degassed

composition of potassium dihydrogen phosphate and acetonitrile in 60:40 v/v ratio was used as

mobile phase for chromatographic analysis. The mobile phase was filtered through a 0.45µm

Millipore filter, before use. UV detection was performed at 263nm. The sample injection volume

was 10µL.

5.3.2. Method Validation

The proposed test method was validated to include requirements of ICH guidelines [60],

in terms of specificity, precision, accuracy, linearity, range and robustness.

5.3.2.1. Specificity

As part of specificity study, the interference of placebo with vildagliptin and metformin

peaks in duplicate preparation of placebo was studied as per the proposed test procedure. The

placebo sample solutions were prepared at various concentrations in the same manner as

described in the sample preparation by taking placebo without active pharmaceutical ingredient

of vildagliptin and metformin. The prepared placebo solution was injected into chromatographic

system and the chromatograms were recorded. There were no interferences due to placebo and

diluents at the retention times of vildagliptin and metformin.

10 µL of blank solution, placebo solution, standard and sample solutions were injected

separately into high performance liquid chromatograph and the chromatograms were recorded

under optimal conditions as shown in Figure 5.3, 5.4 and 5.5 respectively.

5.3.2.3. Precision

5.3.2.3.1. System precision

To evaluate system precision, five replicate samples of vildagliptin and metformin

standard solutions were injected into a high performance liquid chromatographic system and the

chromatograms were recorded. The relative standard deviation, tailing factor and theoretical

plates of vildagliptin and metformin peaks were calculated. The observed relative standard

deviation, tailing factor and theoretical plates for metformin peak were 0.5%, 1.8 and 5444

respectively and for vildagliptin peak they were 0.3%, 1.1 and 14042 respectively. These values

are well within the prescribed acceptance criteria of 2.0%, 2.0, and 3000 for relative standard

deviation, tailing factor and theoretical plates respectively for both metformin and vildagliptin

peaks.

5.3.2.3.2. Intermediate precision

To evaluate intermediate precision, the analysis was carried out by using the

developed method on inter-day and intra-day with two different columns. The observed relative

standard deviation for metformin and vildagliptin on inter-day analysis were 0.5% and 0.4%

respectively and on intra-day analysis were 0.9% and 0.9% respectively. No significant

variability was observed between the results of inter-day and intra- day. The results obtained

from intermediate precision study conclude that the developed method is precise enough to use

for its intended application.

5.3.2.3.3. Methodprecision (repeatability)

The precision of the test method was determined by assaying six samples prepared from

vildagliptin and metformin formulation as per the proposed test procedure, and calculated the

relative standard deviation of the obtained assay results. The obtained method precision results

were found to be satisfactory and are tabulated in table 5.1.

Table 5.1: Method precision results of vildagliptin and metformin.

Preparation No.

Assay results (%)

Vildagliptin Metformin

Precision-1 99.2 99.7






Average 99.3 100.0

% RSD 0.3 0.4

5.3.2.4. Accuracy

To evaluate the accuracy of the proposed method, recovery studies were carried out by

standard addition technique. Solutions were prepared six times at lower (50%) and higher

(150%) level concentrations and in triplicate at 100% (a nominal concentration of about 250µg

mL-1 to 750µg mL-1 for vildagliptin and 2500µg mL-1to 7500µg mL-1 for metformin) of the test

concentration. The solutions were prepared as per the proposed test method at various

concentration levels, injected into the chromatographic system and chromatograms were

recorded. The recoveries were calculated from the obtained results and tabulated in table 5.2 and

5.3.

Table 5.2: Accuracy results of metformin.

Sample

No.

level

(%)

Amount

added

(µg mL-1

)

Amount

found

(µg mL-1

)

Recovery

(%) Statistical Analysis

1 50 2500 2498.8 100.0

Mean* 100.0 2 50 2500 2498.9 100.0

3 50 2500 2500.2 100.0

4 50 2500 2498.6 99.9

% RSD* 0.01 5 50 2500 2498.8 100.0

6 50 2500 2498.4 99.9

1 100 5000 5002.3 100.0

Mean^ 100.2

2 100 5000 5008.8 100.2

3 100 5000 5014.8 100.3 % RSD^ 0.1

1 150 7500 7510.1 100.1

Mean^ 100.2 2 150 7500 7495.0 99.9

3 150 7500 7529.8 100.4

4 150 7500 7506.7 100.1

% RSD* 0.2 5 150 7500 7524.9 100.3

6 150 7500 7528.9 100.4

Overall statistical analysis

Mean$ 100.1

% RSD$ 0.2

Table 5.3: Accuracy results of vildagliptin.

Sample

No.

level

(%)

Amount

added

(µg mL-1

)

Amount

found

(µg mL-1

)

Recovery

(%) Statistical Analysis

1 50 250 251.0 100.4

Mean* 100.2 2 50 250 249.4 99.8

3 50 250 249.2 99.7

4 50 250 251.4 100.6

% RSD* 0.4 5 50 250 250.6 100.2

6 50 250 250.9 100.4

1 100 500 498.8 99.8 Mean^ 99.9

2 100 500 499.9 100.0

3 100 500 499.2 99.8 % RSD^ 0.1

1 150 750 749.9 100.0

Mean^ 100.1 2 150 750 751.9 100.3

3 150 750 751.5 100.2

4 150 750 751.2 100.2

% RSD* 0.1 5 150 750 751.2 100.2

6 150 750 750.5 100.1

Overall statistical analysis

Mean$ 100.1

% RSD$ 0.3

* : For six replicates

^ : For three replicates $ : For fifteen replicates

The results of accuracy as determined by both the calculation methods revealed that, the

average recovery at each level was between 97.0% and 103.0% with RSD at each level ≤ 5%. No

significant difference was seen between the two methods.

5.3.2.5. Linearity

To demonstrate the linearity of detector response for simultaneous assay method of

metformin and vildagliptin, six standard solutions with concentration ranging from 50% to 150%

of metformin and vildagloptin were prepared to cover the concentration of metformin from 2500

to 7500µg mL-1 and for vildagliptin from 250 to 750µg mL-1. The chromatograms were recorded

for these solutions under optimal conditions and the peak areas were measured. Graphs were

plotted between concentration and average peak area. The data were used for statistical analysis

using a linear regression model. The statistical parameters of linear curve (Ca), slope, intercept,

and coefficient of determination values were calculated and shown in Table 5.4. The plotted

graphs were shown in figure 5.6 and 5.7 for metformin and vildagliptin respectively.

Table 5.4: Statistical data of metformin and vildagloptin linearity study

S. No.

Metformin

concentration

(µg mL-1

)

Metformin

peak area

(AU)

Vildagliptin

concentration

(µg mL-1

)

Vildagliptin

peak area (AU)

1

2500 352104 250 106331

2

3750 526230 375 158637

3

5000 701594 500 210444

4

6250 876102 625 263037

5

7500 1057498 750 315605

Coefficient of determination (r2)

0.9999 0.9999

The linearity curve was established by plotting the values of concentration(µg mL-1) on

X-axisand obtained peak areas from chromatographic system (absorbing units) on Y-axis as

determined from linearity test. The obtained coefficient of determination for both metformin and

vildagliptin (r2 = 0.9999) shows that the calibration curve is very much linear in the

concentration range mentioned.

5.3.2.6. Range

To demonstrate the range of concentration of analyte in which the analytical method is

applicable, data from six values of lower and higher concentration solutions of accuracy

preparation were considered. The obtained mean recovery at lowest level and highest level was

found between 97.0% and 103.0% with coefficient of determination (R2) of 0.9999 and the

relative standard deviation for six preparations at each level was found below 2.0% for both

metformin and vildagliptin which shows that the analytical method is more accurate and precise

throughout its range of determination.

5.3.2.7. Robustness

5.3.2.7.1. Study on variable conditions

The robustness of the proposed method was demonstrated by the results obtained in the

study of system suitability parameter by injecting the standard preparation with variable flow

rates (0.8, 1.0 and 1.2 mL minute-1) and at variable column temperatures (40°C and 50°C).

Chromatograms were recorded under variable conditions as mentioned above and plate count

and tailing factor were evaluated for each chromatogram which are presented in Table 5.5. The

observed results of system suitability parameters from the above robustness study were found

well within the acceptance criteria (RSD ≤ 0.3%) which show that the method is robust for the

intended purpose.

Table 5.5: Summary of robustness results

Robustness

Condition Variation

Name of

component USP Tailing

USP Plate

count

As per test Method

- Metformin 1.814 5444

Vildagliptin 1.144 14042

Flow Rate (1 mL minute-1)

- 0.2 mL minute-1 Metformin 1.982 5182


+0.2 mL minute-1 Metformin 1.895 5149


Oven temperature (45°C)

40°C Metformin 1.941 5147


50°C Metformin 1.949 5064


5.3.3. Comparison of the results

Mohammed Abdul et al [51] reported a method for the simultaneous determination of

vildagliptin and metformin in pharmaceutical formulations. No other methods were found for

simultaneous estimation of vildagliptin and metformin. The results of proposed method were

compared with those reported by Mohammed Abdul et al and tabulated the observations in table

5.6.

Table 5.6: Comparison between reported method [51] and present method.

Parameter Reported method [51] Present method Observations

Column Inertsil® CN‐3 column (250 mm length, 4.6mm internal diameter, with 5µm particle size)

Thermo Hypersil ODS (250 mm length x 4.6 mm internal diameter,with 5µm particle size)

C18 columns give good resolution due to their hydrophobic nature. They are easily available, rugged and less expensive when compared with cyano columns.

Mobile phase

Potassium dihydrogen phosphate buffer (pH 4.6) and acetonitrile in the ratio of 15:85v/v.

Potassium dihydrogen phosphate buffer (pH 7.0) and acetonitrile in the ratio of 60:40 v/v.

The composition of mobile phase in the present method gives fast elution with good separation of analyte peaks.

Diluent Methanol Mobile phase was used as diluent.

In the reported method, the mobile phase and diluents were of different composition and hence the complete miscibility is doubtful. In the present method the mobile phase and diluents are one and the same and hence no problem of miscibility.

Flow rate & Pump mode

1.0 mL minute-1 with isocratic mode.

1.0 mL minute-1 with isocratic mode.

Isocratic mode is simple, reliable & gives constant baseline response.

Data Acquisition time

12.5 minutes per injection

5 minutes per injection Less run time reduces solvent consumption, and saves analysis time

Linearity range

Linearity of the method covered from 5µg mL-1 to 200µg mL-1 of vildagliptin and 50µg mL-1 to 2000 µg mL-1 of metformin.

Linearity of the method covered from 250µg mL-1 to 750µg mL-1 of vildagliptin and 2500µg mL-1 to 7500 µg mL-1 of metformin.

Applications will increase with increased range.

5.4 Conclusion

A novel RP-HPLC method was proposed for the simultaneous determination of

vildagliptin and metformin in pharmaceutical finished dosage forms. The analytical method was

validated according to the ICH guidelines which revealed that the method is selective, precise

and accurate. The proposed HPLC method has the ability to separate vildagliptin, metformin

even from excipients found in the tablet dosage form and therefore can be applied to the analysis

of samples at quality control. The method is rapid, direct, specific, accurate, precise, and can be

used for the routine analysis of vildagliptin and metformin drugs in the pharmaceutical finished

dosage form. The method may also be extended to evaluate the active drug substance. The

proposed method is found to be simple and faster compared to the reported method (51) for the

simultaneous determination of vildagliptin and metformin in pharmaceutical dosage forms.

5.5 References:

1. Ahrén, B; Landin-Olsson, M; Jansson, PA; Svensson, M; Holmes, D; Schweizer,

A., Inhibition of dipeptidyl peptidase-4 reduces glycemia, sustains insulin levels, and

reduces glucagon levels in type 2 diabetes. The Journal of Clinical Endocrinology and

Metabolism., 2004; 89 (5); 2078–2084.

2. Mentlein, R; Gallwitz, B; Schmidt WE., Dipeptidyl-peptidase IV hydrolyses gastric

inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine

and is responsible for their degradation in human serum., European journal of biochemistry /

FEBS., 1993; 214 (3): 829–35.

3. Philip E Otterbeck, Mary Ann Banerji, The efficacy and safety of vildagliptin in the

GALIANT trial: chronic kidney disease and other applications, Expert Review of

Endocrinology & Metabolism, 2011, 6 (2), 143-151.

4. Chantal Mathieu and Evy Degrande, Vildagliptin: a new oral treatment for type 2

diabetes mellitus, Vasc Health Risk Manag. 2008; 4(6): 1349–1360.

5. Serge Halimi, Anja Schweizer, Biljana Minic, James Foley, and Sylvie Dejager,

Combination treatment in the management of type 2 diabetes: focus on vildagliptin and

metformin as a single tablet, Vasc Health Risk Manag., 2008; 4(3): 481–492.

6. Fonseca V, Baron M, Shao Q, Dejager S., Sustained efficacy and reduced hypoglycemia

during one year of treatment with vildagliptin added to insulin in patients with type 2

diabetes mellitus. Horm Metab Res, 2008; 40; 427-430.

7. Pi-Sunyer FX, Schweizer A, Mills D, Dejager S., Efficacy and tolerability of vildagliptin

monotherapy in drug-naïve patients with type 2 diabetes., Diabetes Res Clin Pract.,2007;

76; 132–138.

8. Dejager S, Razac S, Foley JE, Schweizer A., Vildagliptin in drug-naïve patients with type

2 diabetes: a 24-week, double-blind, randomized, placebo-controlled, multiple-dose study.,

Horm Metab Res., 2007; 39(3); 218-223.

9. Rosenstock J, Baron MA, Dejager S, Mills D, Schweizer A.,Comparison of vildagliptin

and rosiglitazone monotherapy in patients with type 2 diabetes: a 24-week, double-blind,

randomized trial. Diabetes Care. 2007; 30(2); 217-223.

10. Schweizer A, Couturier A, Foley JE, Dejager S.,Comparison between vildagliptin and

metformin to sustain reductions in HbA(1c) over 1 year in drug-naïve patients with Type 2

diabetes. Diabet Med. 2007; 24(9); 955-961.

11. Pan C, Yang W, Barona JP, Wang Y, Niggli M, Mohideen P, Wang Y, Foley

JE.,Comparison of vildagliptin and acarbose monotherapy in patients with Type 2 diabetes:

a 24-week, double-blind, randomized trial. Diabet Med. 2008;25(4): 435-441.

12. Bosi E, Camisasca RP, Collober C, Rochotte E, Garber AJ.,Effects of vildagliptin on

glucose control over 24 weeks in patients with type 2 diabetes inadequately controlled with

metformin. Diabetes Care. 2007; 30(4); 890-895.

13. Fonseca V, Schweizer A, Albrecht D, Baron MA, Chang I, Dejager S.,Addition of

vildagliptin to insulin improves glycaemic control in type 2 diabetes.,Diabetologia. 2007;

50(6); 1148-1155.

14. He YL.,Clinical pharmacokinetics and pharmacodynamics of vildagliptin., Clin

Pharmacokinet. 2012; 51(3); 147-162.

15. Bailey CJ, Day C., Metformin: its botanical background. Practical Diabetes International.

2004; 21(3); 115–117.

16. Bailey CJ., Metformin: effects on micro and macrovascular complications in type 2

diabetes., Cardiovasc Drugs Ther., 2008; 22;: 215–224.

17. Lord JM, Flight IHK, Norman RJ., Metformin in polycystic ovary syndrome: systematic

review and meta-analysis. BMJ. 2003; 327; 951–953.

18. Marchesini G, Brizi M, Bianchi G, Tomassetti S, Zoli M, Melchionda N., Metformin in

non-alcoholic steatohepatitis., Lancet. 2001; 358(9285):893–894.

19. Ibáñez L, Ong K, Valls C, Marcos MV, Dunger DB, de Zegher F., Metformin treatment

to prevent early puberty in girls with precocious pubarche., J Clin Endocrinol Metab. 2006;

91(8); 2888–2891.

20. Michael Bodmer, Christian Meier, and Christoph R. Meier, Hypoglycemic episodes

were substantially more common among sulfonylurea users than among users of metformin,

Diabetes care,2008; 31(11); 2086-2091.

21. Bolen S, Feldman L, Vassy J, et al. Systematic review: comparative effectiveness and

safety of oral medications for type 2 diabetes mellitus.,Ann Intern Med. 2007;147(6); 386–

399.

22. Radosh L. Drug treatments for polycystic ovary syndrome. Am Fam Physician. 2009; 79(8);

671–676.

23. Tertti K, Ekblad U, Vahlberg T, Rönnemaa T., Comparison of metformin and insulin in

the treatment of gestational diabetes: a retrospective, case-control study., Rev Diabet Stud.

2008; 5(2); 95–101.

24. Rowan JA, Hague WM, Gao W, Battin MR, Moore MP; MiG., Metformin versus insulin

for the treatment of gestational diabetes. N Engl J Med., 2008; 258(19); 2003–2015.

25. Nicholson W, Bolen S, Witkop CT, Neale D, Wilson L, Bass E., Benefits and risks of oral

diabetes agents compared with insulin in women with gestational diabetes: a systematic

review. Obstet Gynecol., 2009; 113(1); 193–205.

26. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD., Metformin and

reduced risk of cancer of 25–37% in diabetic patients. BMJ. 2005; 330: 1304–1305.

27. Libby G, Donnelly LA, Donnan PT, Alessi DR, Morris AD, Evans JM., New users of

metformin are at low risk of incident cancer: a cohort study among people with type 2

diabetes., Diabetes Care. 2009; 32; 1620–1625.

28. Towler MC, Hardie DG., AMP-activated protein kinase in metabolic control and insulin

signaling., Circ Res., 2007; 100(3); 328–341.

29. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber

T, Fujii N, Musi N, Hirshman M, Goodyear L, Moller D., Role of AMP-activated protein

kinase in mechanism of metformin action., J Clin Invest., 2001; 108(8); 1167–1174.

30. Tucker GT, Casey C, Phillips PJ, Connor H, Ward JD, Woods HF., Metformin kinetics

in healthy subjects and in patients with diabetes mellitus., Br J Clin pharmacol., 1981; 12;

235–246.

31. Marathe PH, Wen Y, Norton J, Greene DS, Barbhaiya RH, Wilding IR., Effect of

altered gastric emptying and gastrointestinal motility on metformin absorption. Br J Clin

Pharmacol., 2000; 50; 325–332.

32. Robert F, Fendri S, Hary L, Lacroix C, Andréjak M, Lalau JD., Kinetics of plasma and

erythrocyte metformin after acute administration in healthy subjects., Diabetes Metab.,

2003; 29(3); 279–283.

33. Nathan DM, Buse JB, Davidson MB, et al., Management of hyperglycemia in type 2

diabetes: a consensus algorithm for the initiation and adjustment of therapy: a consensus

statement from the American Diabetes Association and the European Association for the

Study of Diabetes., Diabetes Care., 2006; 29;1963–1972.

34. Bailey CJ, Day C., Fixed-dose single tablet antidiabetic combinations., Diabetes Obes

Metab., 2009; 11(6); 527–533.

35. Guarino E, Nigi L, Patti A, Fondelli C, Dotta F., Combination therapy with metformin

plus vildagliptin in type 2 diabetes mellitus., Expert Opin Pharmacother., 2012; 13(9);

1377-1384.

36. Bosi E, Dotta F, Jia Y, Goodman M.,Vildagliptin plus metformin combination therapy

provides superior glycaemic control to individual monotherapy in treatment-naive patients

with type 2 diabetes mellitus., Diabetes Obes Metab., 2009; 11(5); 506-515.

37. Matthews, D. R., Dejager, S., Ahren, B., Fonseca, V., Ferrannini, E., Couturier, A.,

Foley, J. E. and Zinman, B., Vildagliptin add-on to metformin produces similar efficacy

and reduced hypoglycaemic risk compared with glimepiride, with no weight gain: results

from a 2-year study, Diabetes, Obesity and Metabolism.,2010;12:780-789.

38. Giuseppe Derosa, Pietro D. Ragonesi, Anna Carbone, Elena Fogari, Lucio Bianchi,

Aldo Bonaventura, Davide Romano, Arrigo F.G. Cicero, and Pamela Maffioli.,

Vildagliptin Added to Metformin on β-Cell Function After a Euglycemic Hyperinsulinemic

and Hyperglycemic Clamp in Type 2 Diabetes Patients., Diabetes Technology &

Therapeutics., 2012; 14(6); 475-484.

39. Abd A. Tahrani, Milan K. Piya, Anthony H. Barnett.,Drug evaluation: Vildagliptin-

metformin single-tablet combination, Advances in Therapy, 2009; 26(2); 138-154.

40. Hyun Jeong Jeon and Tae Keun Oh.,Comparison of Vildagliptin-Metformin and

Glimepiride-Metformin Treatments in Type 2 Diabetic Patients, Diabetes Metab J., 2011;

35(5); 529-535.

41. Ahrén, B., Foley, J. E. and Bosi, E., Clinical evidence and mechanistic basis for

vildagliptin's action when added to metformin. Diabetes, Obesity and Metabolism., 2011;

13; 193–203.

42. Claudia Filozof, Sherwyn Schwartz, James E Foley, Effect of vildagliptin as add-on therapy to

a low-dose metformin, World J Diabetes, 2010; 1(1); 19-26.

43. El-Bagary, Ramzia I.; Elkady, Ehab F.; Ayoub, Bassam M.,Spectrophotometric Methods

for the Determination of Sitagliptin and Vildagliptin in Bulk and Dosage Forms, Int J

Biomed Sci.,2011; 7 (1); 55-61.

44. El-Bagary, Ramzia I.; Elkady, Ehab F.; Ayoub, Bassam M.,Liquid Chromatographic

Methods for the Determination of Vildagliptin in the Presence of its Synthetic Intermediate

and the Simultaneous Determination of Pioglitazone Hydrochloride and Metformin

Hydrochloride., Int J Biomed Sci.,2011; 7(3); 201-208.

45. A. B. Pharne, B. Santhakumari, A. S. Ghemud, H. K. Jain, M. J. Kulkarni,

Bioanalytical method development and validation of vildagliptin a novel dipeptidyl

peptidase IV inhibitorby RP-HPLC method, Int J Pharm Pharm Sci, 2012; 4(3), 119-123.

46. Kar M, Choudhury P K,, HPLC method for estimation of metformin hydrochloride in

formulated microspheres and tablet dosage form., Indian J Pharm Sci, 2009; 71; 318-320.

47. Raniah Q. Gabr, Raj S. Padwal and Dion R. Brocks,Determination of Metformin in

Human Plasma and Urine by High-Performance Liquid Chromatography Using Small

Sample Volume and Conventional Octadecyl Silane Column, J Pharm Pharmaceut Sci,

2010; 13(4); 486 – 494.

48. Thamma Narendra kumar, Kota Chandra Mohan Rao, R. Sreenivasulu, Dr.NSV Raju

and Viswanath Reddy Pyreddy, Novel Rp-Hplc Method For The estimation Of Metformin

Hydrochloride In Pharmaceutical Dosage Forms, International Journal of Science

Innovations and Discoveries., 2011; 1 (3); 395-421.

49. Valentina Porta, Simone Grigoleto Schramm, Eunice Kazue Kano, Eunice Emiko

Koono, Yara Popst Armando, Kazuo Fukuda, Cristina Helena dos Reis Serra,HPLC-

UV determination of metformin in human plasma for application in pharmacokinetics and

bioequivalence studies, J. Pharm.Biomed.Anal., 2008; 46(1); 143‐147.

50. K.Pushpa Latha And D.Ramachandran, Method development and validation for the

simultaneous estimation of vildagliptin and metformin in tablet dosage form by RP-HPLC,

Int J Pharm Pharm Sci, 2013; 5(1), 459-463.

51. Mohammad Abdul‐Azim Mohammad, Ehab Farouk Elkady and Marwa AhmedFouad.,

Development and validationof areversed‐phase column

liquidchromatographicmethodforsimultaneous determination of two novel gliptins in their

binary mixtures with Metformin., European Journal of Chemistry., 2012; 3(2); 152‐155.

52. Usharani Gundala, Chandra Shekar Bhuvanagiri, Devanna Nayakanti, Simultaneous

Estimation of Vildagliptin and Metformin in Bulk and Pharmaceutical Formulations by UV

Spectrophotometry, Am. J. PharmTech Res, 2013;3(1), 338-345.

53. G.Alekya, Naira Nayeem, T Mahati, RP-HPLC Method Development and Validation of

Metformin and Vildagliptin in Bulk and Its Pharmaceutical Dosage form and their Bio-

Analytical Studies, Am. J. PharmTech Res, 2013;3(4), 358-369.

54. P. G. Shelke, A. P. Dewani, R. L. Bakal, S. N. Vasu, A.S. Tripathi and A.V. Chandewar, A

Validated RP-HPLC Method For Simultaneous Determination Of Metformin HCl And

Vildagliptin In Pharmaceutical Formulation, International Journal of Advances in

Pharmaceutical Analysis, 2013; 3(2), 37-41.

55. BM Ishaq, KV Prakash, GK Mohan, RP-HPLC method for simultaneous estimation of

metformin and vildagliptin in bulk and its tablet formulation, Global Trends Pharm. Sci.,

2012; 3(3), 747-754.

56. Hitesh P. Inamdar, Ashok A. Mhaske, Shirish P. Sahastrabudhe, A revised RP-HPLC method

for simultaneous determination of vildagliptin and pioglitazone hcl – application to

commercially available drug products, International journal of pharmaceutical sciences and

research, 2013; 4(2): 847-855.

57. Vinay Pandit, Roopa S. Pai, Kshama Devi, Gurinder Singh, Satya Narayana, and Sarasija

Suresh, Development and validation of the liquid chromatographic method for

simultaneous estimation of metformin, pioglitazone, and glimepiride in pharmaceutical

dosage forms, Pharm. Methods, 2012; 3(1), 9-13.

58. Sunil R. Dhaneshwar, Janaki V. Salunkhe and Vidhya K. Bhusari, Validated HPTLC Method

for Simultaneous Estimation of Metformin Hydrochloride, Atorvastatin and Glimepiride in

Bulk Drug and Formulation, J Anal Bioanal Tech, 2010; 1, 109.

59. Gadapa Nirupa and Upendra M. Tripathi, “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, 2013, Article ID

726235, 8 pages.

60. International Conference on Harmonization of technical requirements for registration of

pharmaceuticals for human use, ICH harmonized tripartite guideline, validation of analytical

procedures: Text and methodology Q2 (R1), step 4 2005.

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Chapter-5 Simultaneous estimation of vildagliptin and ...shodhganga.inflibnet.ac.in/bitstream/10603/20011/11... · metformin treatment fails to achieve glycemic goals [33]. When used