STABILITY INDICATING METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF ANTI DIABETIC IN FIXED DOSE COMBINATIONS

IDENTIFICATION, CHARACTERIZATION AND QUANTIFICATION OF HETEROCYCLIC
COMPOUNDS IN BIOLOGICAL MATRICESAND THEIR APPLICATION TO
PHARMACOKINETIC STUDIES
A THESIS
Submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD
in the partial fulfillment of the requirements for
the award of the degree of
BACHELOR OF PHARMACY
By
D. VIJAYA BHARATHI [Reg. No. 0503PH0226]
Under the Guidance of
M. Pharm
Assistant Professor
GLAND INSTITUTE OF PHARMACEUTICAL SCIENCES

STABILITY INDICATING METHOD DEVELOPMENT AND
VALIDATION FOR SIMULTANEOUS ESTIMATION OF
ANTI-DIABETIC DRUGS IN FIXED-DOSE COMBINATIONS
INTRODUCTION
METHOD DEVELOPMENT
Analytical Method Development is required for New process and reactions, New molecules, Active
ingredients (Macro analysis), Residues (Microanalysis), Impurity Profiling, Components of interest in
different matrices. An analytical methodology consists of Techniques, method, procedure, and protocol.
Basic criteria for new method development of drug analysis:
 The drug or drug combination may not be official in any pharmacopoeias,
 A proper analytical procedure for the drug may not be available in the literature due to patent
regulations,
 Analytical methods may not be available for the drug in the form of a formulation due to the
interference caused by the formulation excipients.
 Analytical methods for the quantitation of the drug in biological fluids may not be available.
 Analytical methods for a drug in combination with other drugs may not be available.
 The existing analytical procedures may require expensive reagents and solvents. It may also
involve cumbersome extraction and separation procedures and these may not be reliable.
METHOD VALIDATION
Method validation is the process used to confirm that the analytical procedure employed for a specific
test is suitable for its intended use.
Method validation is:
 Necessary to judge the quality, reliability and consistency of analytical results; it is an integral
part of any good analytical practice.
 Necessary for achieving acceptance of products by the international agencies.
 Mandatory requirement purpose for accreditation as per ISO 17025 guidelines.
 Mandatory requirement for registration of any pharmaceutical product or pesticide formulation.

 A regulatory requirement that is of great importance in the pharmaceutical industry, because they
ensure the quality of the product that is made [1].
Analytical methods need to be validated or revalidated:
 before their introduction into routine use;
 whenever the conditions change for which the method has been validated (e.g., an instrument with
different characteristics or samples with a different matrix); and
 whenever the method is changed and the change is outside the original scope of the method.
For validation the developed method is subjected to Precision /Reproducibility, Accuracy, Linearity,
Specificity / Selectivity, Limit of detection, Limit of quantitation, Robustness / Ruggedness.
The present research is envisaged on new analytical method development and validation for simultaneous
estimation of Anti-diabetic fixed-dose combination drugs[3].
REVIEW OF RELATED LITERATURE
The Combination Therapy of different mechanism of action of drugs plays an important role in the
measurement of Type II Diabetes Mellitus. The effect of diabetes develops specific complications of
retinopathy, nephropathy, neuropathy, cardiac vascular diseases and cerebro vascular diseases.
Combination therapy is beneficial for various diseases over monotherapy. The oral hypoglycemic drug
combination therapy is most suitable for chronic disorder. Sulfonyl ureas, Alpha glucosidase inhibitors,
Biguanides, Meglitinides and Thiazolidinediones are important in Combination Therapy. Bilayer tablet is
suitable for sequential release of two drugs in combination, in which one layer is initial dose as
immediate release and second layer is maintenance dose. This article explains the dual therapy of bilayer
tablet and combination of anti-diabetic drugs which improves the patient compliance and provides
synergistic effect[4].

Metformin Hydrochloride:
Metformin is an oral anti-diabetic drug in the biguanide class. It is chemically N, N
dimethylimidodicarbonimidic diamide hydrochloride (1, 1 dimethyl biguanide hydrochloride) (Fig .2) It
lowers blood glucose concentrations in type 2 diabetes without causing overt hypoglycemia. Metformin
is also frequently described as an insulin sensitizer leading to reduction in insulin resistance and
significant reduction of plasma fasting insulin level. The improvement in insulin sensitivity by metformin
could be ascribed to its positive effects on insulin receptor expression and tyrosine kinase activity [10].
Metformin reduces hepatic glucose production. Metformin is small highly polar compound (pKa=2.8,
11.5) so it has a great solubility in water and poor solubility in lipids so it is very difficult to extract it
from the aqueous plasma matrix. HPLC methods for the determination of metformin in human plasma
include ion-exchange, ion-pair or normal –phase extraction [11-12]. Metformin inhabits hepatic
gluconeogenesis in mice independently of the LKB1/AMPK pathway via decrease in hepatic energy state
[6-13].
Empagliflozin:
Empagliflozin chemically,(1-chloro-4-[b-D-glucopyranos-1-yl]-2-[4-([S]-tetrahydrofuran-3-yl-oxy)
benzyl]-benzene (Fig.1) is an orally administered selective sodium glucose cotransporter-2 (SGLT-2)
inhibitor, which lowers blood glucose in people with type 2 diabetes by blocking the reabsorption of
glucose in the kidneys and promoting excretion of excess glucose in the urine [1-6]. In patients with type

2 diabetes and hyperglycaemia a higher amount of glucose is filtered and reabsorbed. Empagliflozin
improves glycaemic control in patients with type 2 diabetes by reducing renal glucose reabsorption. The
amount of glucose removed by the kidney through this glucuretic mechanism is dependent on blood
glucose concentration and GFR. Inhibition of SGLT2 in patients with type 2 diabetes and
hyperglycaemia leads to excess glucose excretion in the urine[5,14-16]
Sitagliptin & Saxagliptin:
Sitagliptin phosphate monohydrate chemically (3R)-3-amino-1-[3-(trifluoromethyl)-5,6-
dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one phosphate hydrate is
an oral anti-diabetic, Saxagliptin chemically, (1S,3S,5S)-2-[(2S)-2-Amino-2-(3hydroxy tricycle
[3.3.1.13,7]dec-1-yl) acetyl]-2-azabicyclo[3.1.0] hexane-3-carbonitrile.Saxagliptin is an orally active
hypoglycemic of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs that was being
introduced after Sitagliptin. Moreover Sitagliptin and Saxagiptin are used for the improvement of
glycemic control in patients with type II diabetes mellitus as monotherapy or combination therapy with
metformin or a peroxisome proliferator activated receptor gamma (PPAR) agonist (e.g.,
thiazolidinediones) when the single agent does not provide adequate glycemic control. Rising demand of
DPP-4 class inhibitor drug in pharmaceutical market, it is required to develop a new cost-effective, fast
and precise analytical LC technique for the estimation of drug in bulk as well as pharmaceutical dosage
forms. Literature review indicate that few simultaneous LC methods have been reported for analysis of
DPP-4 inhibitor class drugs such as Sitagliptin with Metformin and for Metformin with Saxagliptin
without stability study but there is not at all any multicomponent techniques were developed or reported
for the estimation on above mentioned drugs in bulk or pharmaceutical dosage form. Additionally,
existing simultaneous methods were less cost-effective and more time consuming not fit for the
multicomponent estimation. Hence the main goal of the project is to develop single cost-effective LC
method for the analysis of metformin, saxagliptin and sitagliptin that wasn't developed so far, hence it
may apply with success for the estimation of 2 totally different pharmaceutical combos[17-23].
Linagliptin:
Linagliptin chemically {1H-Purine-2, 6-dione, 8-[(3R)-3-amino-1-piperidinyl]-7-(2-butyn-1-yl)-3,7-
dihydro-3-methyl-1-[(4-methyl-2- quinazolinyl) methyl]}is a DPP-4 inhibitor used for the treatment of
type II diabetes. DPP-4 (dipeptidyl peptidase- 4) is an enzyme that degrades the hormones, Glucagon like

peptide-1 (GLP-1) and Glucose dependent Insulinotropic polypeptide (GIP). Both GLP-1 and GIP
enhance insulin biosynthesis and secretion from pancreatic beta cells in the presence of normal and
elevated blood glucose levels. GLP-1 also reduces glucagon secretion from pancreatic alpha cells,
resulting in a reduction in hepatic glucose output. Thus, linagliptin stimulates the release of insulin in a
glucose-dependent manner and decreases the levels of glucagon by inhibiting DPP-4 and increasing the
levels of GLP-1 and GIP . Linagliptin is indicated as an adjunct to diet and exercise to improve glycemic
control in patients with type 2 diabetes [24-26].
Vildagliptin :
Vildagliptin (previously LAF237, trade names Galvus, Zomelis,) is an oral anti-hyperglycemic agent
(anti-diabetic drug) of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. Vildagliptin
inhibits the inactivation of GLP-1[2][3] and GIP[3] by DPP-4, 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 hyperglycemia in type 2 diabetes
mellitus[27].
Glimepiride:
Glimepiride chemically, shown in figure 1, is 3-ethyl-4- methyl-N-[2-[4-[(4-methylcyclohexyl)
carbamoylsulfamoyl] phenyl] ethyl]-2-oxo-5H-pyrrole-1-carboxamide is a third generation sulfonylurea
derivative which is commonly used in the treatment of non-insulin dependent Type 2 diabetes
mellitus.1,2 It is a medium –to-long acting anti-diabetic drug which acts as a secretagogue [28-31].
Gliclazide:
Gliclazide (1-(3-azabicyclo [3.3.0] oct- 3- yl) - 3- ptolylsulfonylurea or 1-(hexahydrocyclopenta
[c]pyrrol-2 (1H)-yl) - 3- (p-tolylsulfonyl) urea is an oral hypoglycemic agent used in the treatment of
type-II diabetes mellitus (Fig 1). It belongs to the sulfonylurea class which act by stimulating β cells of
the pancreas to release insulin. It reduces blood glucose levels by correcting both defective insulin
secretion and peripheral insulin resistance, increasing the sensitivity of ß- cells to glucose, decreasing
hepatic glucose production, and increasing glucose clearance. It also has anti-platelet adhesive activity
and reduces levels of free radicals, thereby preventing vascular complications. It also has been reported to
reduce plasma cholesterol and triglyceride levels after repeated administration [32].

Pioglitazone Hydrochloride:
The active moiety of pioglitazone hydrochloride (PIO) (5-[[4-[2-(5-ethylpyridin-2-
yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione) is a thiazolidinedione, a potent and highly selective
agonist for the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR-γ). PPARs are
found in tissues like adipose tissue, skeletal muscle and liver, which are critical to insulin action.
Activation of PPAR-γ modulates the transcription of a number of insulin-responsive genes involved in
the control of glucose and lipid metabolism and It is not chemically or functionally related to the alpha-
glucosidase inhibitors, the biguanides, or the sulfonylureas. It addresses main pathophysiological defect
i.e., insulin resistance, so it is used alone or in combination with insulin, metformin, or a sulfonylureas
(glimepride and glibenclamide) as an agent to treat diabetes. PIO reduces peripheral and hepatic
resistance to insulin, resulting in increased insulin dependent glucose disposal and decreased hepatic
‐
glucose output[33-37].
OBJECTIVES OF THE STUDY:
The present investigation is proposed and focused with the objective of:
 Selecting of latest fixed dose combinations of anti diabetic products.
 Selecting of the marketed formulations which are going to expire the patents in a very short span
of time.
 Developing a simple, precise and accurate method for estimating the drug content in the marketed
formulations using HPLC.
 Demonstration of specificity by applying stress treatment to drugs in acidic, basic, oxidative and
photo-irradiation conditions.
 Validating the developed methods as per ICH guide lines.
 Determining the drug content in the marketed formulations by using the validated methods.
 Comparision of innovator formulations with the generic formulations if any generic formulation
is introduced in to the market.
HYPOTHESIS
Though some methods are available for determination of fixed dose anti diabetic drugs, there are no
sufficient methods for their estimation as the drugs and RLD’s are very costly, not easily available and a
lot of care should be taken while handling the drugs. Therefore, I assume that the developed methods will

1. Be simple, accurate, precise, rugged & cost effective.
2. Stable with out any major deviations.
3. Have a good & easy extraction procedure for estimation of drug in their formulations.
4. Easy to validate with out any complications
5. Show the degradation of the formulated drug by some specific degradation material.
Preliminary studies in this regard are undertaken in our lab. We have started procuring and developing
methods for the determination of drugs in pharmaceutical formulations.
RESEARCH METHODOLOGY
INITIAL PARAMETERS IN METHOD DEVELOPMENT
 Checking the solubility for API in suitable solvent.
 Selection of proper diluent for extracting the API from dosage form.
 Checking the absorbance maximum of the API.
 Selecting proper concentration of the drug for standard preparation
 Checking of pKa of the drug by literature review.
 Selection of mode of separation based on the nature of the drug.
 Selection of stationary phase
 Selection of mobile phase, buffer, if any and its strength & pH of the buffer
 Selection of detector
 Mobile phase composition
 Selection of detector
 Forced Degradation Studies
THE DEVELOPED METHOD WILL BE VALIDATED ACCORDING TO THE ICH GUIDELINES Q2(R1)
FOR THE FOLLOWING PARAMETERS:
System Suitability Precision Accuracy
Specificity Limit of detection and quantification Linearity and Range
Recovery Stability Studies Robustness
Forced degradation studies

SAMPLING:
This stage focuses on the selection of the sample solvent (for extraction) and the proper sample
preparation procedures. Investigate the effect of sample solvents of different % organic, pH,
extraction volume and extraction procedure on accuracy, precision, sensitivity (LOQ) and the changes in
the chromatography (e.g., peak shape, resolution). This will ensure that there will not be any
compatibility issues between the sample solution and the HPLC or UPLC conditions.4
TOOLS REQUIRED & AVAILABLE FOR CARRYING RESEARCH:
S.No. Name of the Instrument Make Model
1 HPLC Waters with Empower software Alliance 2695 with PDA
2 UPLC Waters with Empower software Aquity with PDA
3 GC YOUNGLIN 6000 SERIES
4 FTIR Spectrophotometer Shimadzu 8400S
5 UV Visible spectrophotometer LABINDIA UV3000+
6 Electronic Balance Afcoset ------
7 Digital pH Meter ADWA ------
8 Magnetic stirrer Remi Equipments 2MLH
9 Centrifuge Remi Equipments 2MLH
10 Hot Air Oven Thermo 20L-OV
11 Refrigerator LG ------
12 Tablet Disintegration Tester LABINDIA DL-08T
13 HPLC & UPLC columns Waters, Hypersil & Inertsil ------
DELIMITATIONS:
 Instability of drugs may also lead to problems.
 Fixed dose Anti-diabetic drugs are costly and difficult to procure as they are manufactured by
very few companies.
 Special care should be taken while handling the drugs as they lead to serious disorders.

ANALYSIS AND INTERPRETATION OF DATA
The sample or solute is analyzed quantitatively by either peak height or peak area measurements. Peak
areas are proportional to the amount of constant rate. Peak heights are proportional to the amount of
material only when peak width are constant and are strongly affected by the sample injection techniques.
Once the peak height or the peak areas are measured, there are five principle evaluation methods for
quantifying the solute.
a) Calibration by Standards
Calibration curves for each component are prepared from pure standards, using identical injection
volumes of operating conditions for standards and samples. The concentration of solute is read from its
curve if the curve is linear.
b) Internal Standard Method
In this technique a known quantity of the internal standard is chromatographed and area vs
concentration is ascertained. Then a quantity of the internal standard is added to the raw sample prior to
any sample pretreatment or separation operations.
The peak area of the standard in the sample run is compared with the peak area when the standard
is run separately. This ratio serves as a correction factor for variation in sample size, for losses in any
preliminary pretreatment operations, or for incomplete elution of the sample. The material selected for
the internal standard must be completely resolved from adjacent sample components, must not interfere
with the sample components and must never be present in samples.
c) Area Normalization
The technique is often used for the sample having identical components. It is used to evaluate the
absolute purity of the sample. The procedures are to total up the areas under all peaks and then calculate
the percentage of the total area that is contributed by the compound of interest. For this method the entire
sample must be eluted, all components must be separated and each peak must be completely resolved.

d) Standard Addition Method
If only few samples are to be chromatographed, it is possible to employ the method of standard
addition(s). The chromatogram of the unknown is recorded, then a known amount of analyte(s) is added
and the chromatogram is repeated using same reagents, instruments and other conditions. From the
increase in the peak area (or peak height), the original concentration can be computed by interpolation.
The detector response must be a linear function of analyte concentration and yield no signal
at zero concentration of the analyte. Sufficient time must elapse between addition of the standard and
actual analysis to allow equilibrium of added standard with any matrix interferant.
e) External Standard method
It employs a separate injection of a fixed volume of sample and standard solution. The peaks are
integrated and concentration is calculated.
NEED AND SCOPE OF THE STUDY
The number of drugs introduced into the market is increasing every year. These drugs may be either new
entities or partial structural modification of the existing one. Very often there is a time lag from the date
of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens
because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new
toxicities (resulting in their withdrawal from the market), development of patient resistance and
introduction of better drugs by competitors. Under these conditions, standards and analytical procedures
for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop
newer analytical methods for such drugs.
The scope of developing and validating analytical methods is to ensure a suitable method for a
particular analyte more specific, accurate and precise. The main objective for that is to improve the
conditions and parameters, which should be followed in the development and validation.
A survey of literature reveals that very few good analytical methods are available for fixed dose
anti-diabetic drugs. Even though very few methods of estimation of above drugs are available, many of
them suffer from one disadvantage or the other, such as low sensitivity, lack of selectivity and simplicity
etc.

Analysis of fixed-dose combinations of anti-diabetic drugs in formulated and unformulated
samples demand a highly specific and rapid method as many have serious stability problems. HPLC or
UPLC techniques can provide a valuable tool for generating highly pure preparations for characterizing
the antifungal activities.[3]
The existing physicochemical methods are inadequate to meet the requirements; hence it is
proposed to improve the existing methods and to develop new methods for the assay of fixed dose Anti-
diabetic drugs in pharmaceutical dosage forms adapting different available analytical techniques like
HPLC.
SCOPE AND SIGNIFICANCE OF THE STUDY:
Today pharmaceutical analysis entails much more than the analysis of active pharmaceutical ingredients
or the formulated product. The pharmaceutical industry is under increased scrutiny from the government
and the public interested groups to deliver to market safe, efficacious product that fulfill unmet medical
needs. The pharmaceutical analyst plays a major rule in assuring identity, safety, efficacy, purity, and
quality of a drug product. The need for pharmaceutical analysis is driven largely by regulatory
requirements. The commonly used tests of pharmaceutical analysis generally entail compendia testing
method development, setting specifications, and method validation. Analytical testing is one of the more
interesting ways for scientists to take part in quality process by providing actual data on the identity,
content and purity of the drug products. New methods are now being development with a great deal of
consideration to worldwide harmonization. As a result, new products can be assured to have comparable
quality and can be brought to international markets faster.
Pharmaceutical analysis occupies a pivotal role in statuary certification of drugs and their
formulations either by the industry or by the regulatory authorities. In industry, the quality assurance and
quality control departments play major role in bringing out a safe and effective drug or dosage form. The
current good manufacturing practices (cGMP) and the Food Drug Administration (FDA) guidelines insist
for adoption of sound methods of analysis with greater sensitivity and reproducibility. Therefore, the
complexity of problems encountered in pharmaceutical analysis with the importance of achieving the
selectivity, speed, low cost, simplicity, sensitivity, specificity, precision and accuracy in estimation of
drugs.

EXPECTED OUTCOMES OF THE STUDY
To develop a method which is rapid, selective, accurate, rugged, precise and which involves a simple
extraction procedure. Moreover, it should involve lower solvent consumption and lead to a cost effective
and represent a good procedure for determination of drugs in bulk & in pharmaceutical dosage forms.
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