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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
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
ype 2 diabetes mellitus. It is used to treat type 2 or non-insulin dependent
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 of two incretin hormones found in the body,
1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP).
These hormones are normally produced naturally by the body in response to food intake. Their
d sugar (glucose) levels.GLP-1 and GIP have two main actions that
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
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
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
insulin dependent
diabetes mellitus (NIDDM). The chemical structure of vildagliptin is as shown in Figure 5.1.
of two incretin hormones found in the body,
dependent insulinotropic peptide (GIP).
These hormones are normally produced naturally by the body in response to food intake. Their
1 and GIP have two main actions that
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
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
early treatment with metformin is associated with reduced cardiovascular morbidity and total
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].
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.
, 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
anovulation, but also on insulin resistance, hirsutism, and obesity often associated
polycystic ovary syndrome (PCOS) [22]. Several observational studies and randomized,
controlled trials have found that metformin is as effective and safe as insulin for the management
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
early treatment with metformin is associated with reduced cardiovascular morbidity and total
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
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. Its
and its chemical structure is as
line treatment option because it has positive effects not only on
anovulation, but also on insulin resistance, hirsutism, and obesity often associated
and randomized,
controlled trials have found that metformin is as effective and safe as insulin for the management
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.
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 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.
Flow rate: 1.0 mL minute-1
Injection Volume: 20 µL
Data acquisition time: 20 minutes
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.
Column: Thermo Hypersil ODS column, 250mm length, 4.6mm internal diameter and
porous silica packed with particle size of 5µm.
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.
Flow rate: 1.0 mL minute-1
Injection volume: 10 µL
Data acquisition time: 5 minutes
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
Precision-2 98.7 99.5
Precision-3 99.1 100.2
Precision-4 99.5 99.6
Precision-5 99.7 100.3
Precision-6 99.2 100.5
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
Vildagliptin 1.150 14868
+0.2 mL minute-1 Metformin 1.895 5149
Vildagliptin 1.130 14345
Oven temperature (45°C)
40°C Metformin 1.941 5147
Vildagliptin 1.150 14868
50°C Metformin 1.949 5064
Vildagliptin 1.130 14778
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.
Recommended