“ FORCED DEGRADATION OF TABLET DOSAGE FORM OF VILDAGLIPTIN AND

LINAGLIPTIN”
A PRESENTATION TO
SAVITRIBAI PHULE PUNE UNIVERSITY, PUNE
PROJECT WORK COURSE
(BP813PW)
IN THE FACULTY OF SCIENCE AND TECHNOLOGY
FINAL YEAR B. PHARMACY,
SEMESTER- VIII
PRESENTED BY
MR. KUYATE NIRUPAM VASANT
FINAL YEAR B. PHARM., ROLL NO. 35
UNDER GUIDANCE OF
Dr. L. A. KAWALE,
M. Pharm., Ph. D

AT
MARATHA VIDYA PRASARAK SAMAJ’s
COLLEGE OF PHARMACY
GANGAPUR ROAD, NASHIK-422 002 (M.S)
ACADEMIC YEAR: 2023-2024
Contents:
• Abstract
• Introduction
• Aim and Objectives
• Experimental Details
• Results
• Conclusion
• References
Abstract:
• Forced degradation studies are imperative in elucidating the
stability and degradation pathways of pharmaceutical
compounds. This abstract delves into the forced degradation
studies conducted on Vildagliptin and Linagliptin, two important
dipeptidyl peptidase-4 (DPP4) inhibitors widely used in the
treatment of type 2 diabetes mellitus. Various stress conditions,
including thermal, oxidative, photolytic, and hydrolytic stress,
can be employed to induce degradation under controlled
conditions. In this experiment mainly alkali, acidic and oxidative
degradation have been studied. Many analytical techniques
such as High-performance liquid chromatography (HPLC)
coupled with Mass Spectroscopy can be utilized to characterize
degradation products and elucidate degradation pathways. The
findings reveal potential degradation pathways and highlight
the stability profiles of Vildagliptin and Linagliptin under stress
conditions. These studies provide crucial insights for the
development of robust formulations and regulatory compliance
in pharmaceutical drug development
Introduction
• Forced degradation studies involve subjecting a drug substance or product to stress
conditions like heat, light, humidity, and pH to accelerate degradation. They help
identify potential degradation pathways and degradation products, aiding in the
development of stability-indicating methods for quality control.

• These studies are crucial for assessing the stability of pharmaceuticals and ensuring
their efficacy and safety throughout their shelf life.

• Stability testing provides evidence that the quality of drug substance or drug product
changes with time under the influence of various environmental conditions such as
temperature, relative humidity etc.
• Forced degradation or stress degradation is a process whereby the natural degradation rate of a product is
increased by the application of additional stress

Flow chart of Forced Degradation

• Types of Forced Degradation
a) Acid and Alkali Hydrolysis:
The hydrolytic degradation of a new drug in acidic and alkaline condition can be studied
in
0.1N HCL/ 0.1N NaOH. If reasonable degradation is seen, testing can be stopped at this
point.
However, in case no degradation is seen under these conditions’ degradation should be
tried
in HCL/NaOH of higher strength & for longer duration of time. Alternatively, if total
degradation is seen after subjecting the drugs to initial condition, acid/alkali strength can
be
decreased along with decrease in reaction temperature.
b) Oxidative Degradation:
To test for oxidation, it is suggested to use hydrogen peroxide in the concentration range
of 3
to 50%. In some drugs, extensive degradation is seen when exposed to 3% of hydrogen
peroxide for very shorter time period at room temperature. In other cases, exposure to
high concentration of hydrogen peroxide, even under extreme condition does not cause
any significant degradation.
c) Photolytic Degradation
UV light: The photolytic studies should be carried out by exposure to light using either a
combination of cool white & UV fluorescent lamp. Exposure energy should be minimum
of
1.2 million lux hrs fluorescent light and if decomposition is not seen the intensity should
be
increased by 5 times. In case still no decomposition takes place, the drug can be
declared photo
stable
d) Heat Degradation
Heating the drug powder at higher temperature in oven can carry out stress testing for
dry heat
degradation. The heating time can be increased up to 12 hours if no sufficient
degradation is seen in initial studies
UV – Visible Spectrophotometric Method.
• UV spectrophotometry is a technique used to analyze the
absorption, transmission, and reflection of light in the
ultraviolet-visible range of the electromagnetic spectrum
(typically 190 to 1100 nm). It's commonly employed in
various fields including chemistry, biochemistry,
pharmaceuticals, and environmental science.
• In practice, a spectrophotometer measures how much light
a sample absorbs at different wavelengths. This data can
provide information about the sample's concentration,
purity, or chemical structure, as different molecules absorb
light at specific wavelengths. UV-visible spectroscopy is
widely used for quantitative analysis, such as determining
the concentration of a substance in a solution, and
qualitative analysis, like identifying functional groups or
chemical bonding in a compound.
• Instrumentation: A typical UV-visible spectrophotometer consists of a light source
(usually a deuterium and tungsten lamp for UV and visible regions, respectively), a
monochromator to select the desired wavelength, a sample holder, and a detector
(photomultiplier tube or photodiode array) to measure the intensity of transmitted or
absorbed light.

• Sample Preparation: Samples are typically dissolved in a suitable solvent and placed in a
cuvette, which is then inserted into the spectrophotometer. The solvent used should not
absorb significantly in the UV-visible range.

• Measurement: The spectrophotometer measures the intensity of light passing through the
sample at different wavelengths. A spectrum is generated, showing the absorbance or
transmittance of the sample as a function of wavelength.

• Analysis: The obtained spectrum can provide information about the structure,
concentration, and chemical environment of the sample. Quantitative analysis can be
performed by comparing the absorbance of the sample at a specific wavelength to a
standard curve or by using Beer-Lambert law.
There are two laws which govern the absorption of light by the molecules.
These are (1) Lambert's law and (2) Beer’s law.
Lambert law states that –
“When a beam of monochromatic radiation passes through a homogeneous absorbing medium, the
rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to the
intensity of the incident radiation”.
Beer’s law states that –
“When beam of monochromatic radiation is passed through a solution of an absorbing substance, the
rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to
the intensity of incident radiation as well as the concentration of the solution.
Overall, UV-visible spectroscopy is a powerful and versatile technique that provides valuable
insights into the properties and behavior of molecules in solution.
Drug Profile:
1. Vildagliptin

a) Mechanism of Action:
• Vildagliptin works to improve glycemic
control in type II diabetes mellitus by
enhancing the glucose sensitivity of
beta-cells (β-cells) in pancreatic islets
and promoting glucose dependent
insulin secretion. Increased GLP-1
levels lead to enhanced sensitivity of
alpha cells to glucose, promoting
glucagon secretion.
• Vildagliptin causes an increase in the insulin to glucagon ratio by increasing incretin
hormone levels: this results in a decrease in fasting and postprandial hepatic glucose
production.

• Vildagliptin does not affect gastric emptying. It also has no effects on insulin secretion or
blood glucose levels in individuals with normal glycemic control.

b) Uses:

• Vildagliptin is indicated in the treatment of type II diabetes mellitus in adults.

• As monotherapy, vildagliptin is indicated in adults inadequately controlled by diet and exercise

alone and for whom metformin is inappropriate due to contraindications or intolerance.

• It is also indicated as dual therapy in combination with metformin, a sulphonylurea, or a

thiazolidinedione in adult patients with insufficient glycemic control despite maximal

tolerated dose of monotherapy.

2. Linagliptin

a) Mechanism of Action:

Linagliptin is an inhibitor of DPP-4, an enzyme that degrades the incretin hormones

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

Thus, linagliptin increases the concentrations of active incretin hormones, stimulating the

release of insulin in a glucose-dependent manner and decreasing the levels of glucagon in the

circulation.
b) Uses:

• Linagliptin is used with a proper diet and exercise program and possibly with other

medications to control high blood sugar. It is used by people with type 2 diabetes.

• Linagliptin is a diabetes drug that works by increasing levels of natural substances called

incretins. Incretins help to control blood sugar by increasing insulin release, especially after a

meal. They also decrease the amount of sugar your liver makes
• Aim & Objectives
• The main aim of the work was to determine and evaluate the change in the structure and composition
of Vildagliptin and Linagliptin when they were forcefully degraded under basic, acidic and oxidative
conditions.

• Objectives:

1. To establish degradation pathways of drug substances and drug products.

2. To determine the intrinsic stability of a drug substance in formulation.

3. To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis or

photolysis of the drug substance and drug product.

4. To establish the stability indicating nature of a developed method.

5. To understand the chemical properties of drug molecules.

Experimental Details
Material and Instruments.

• Specification of drug standards:

Sr. no. Name of the drug Name of the Strength Manufactur Supplied by Quantity
tablet ed
formulation by
1. Vildagliptin Gliptagreat 50mg Mankind Health 2 strips of
Pharma. Ltd Planet 15 tablets
Pharma and each.

2. Lifestyle
Linagliptin Linapil 5mg Alkem 3 strips of
Store,
Health 10 tablets
Nashik.
Science. Ltd each.
• Reagents and Chemicals:

Sr. no. Reagents and Chemicals Details

1. 0.1N NaOH AR grade
2. 0.1N HCL AR grade
3. 3% H2O2 AR grade
4. Ethanol AR grade
5. Distilled water AR grade

 INSTRUMENTS:
1. UV SPECTROPHOTOMETER
Make: Shimadzu
Model: UV-2501PC
2. ANALYTICAL BALANCE:
Make: Shimadzu
Model: ATX224
• Experimental Methodology:

A) UV SPECTROSCOPY METHOD DEVELOPMENT:

The parameters for the development were as follows:

1. Selection of solvent for Vildagliptin and Linagliptin.

2. Preparation of standard solution of Vildagliptin and Linagliptin.

3. Determination of detection wavelength.

1. Selection of Solvent:

•The ideal properties of the solvent to be used in UV Spectrophotometry include:

• Drugs that are Vildagliptin and Linagliptin should show solubility and stability in the selected
solvent.
• Both the drugs that are Vildagliptin and Linagliptin should obey the Beer-Lambert’s law over an
appropriate range of analytical considerations.

• After taking into consideration the above factors, water was selected as a solvent for analysis of
Vildagliptin and Ethanol was selected as a solvent for analysis of Linagliptin

2. Preparation of 100ppm solutions of Vildagliptin and Linagliptin (Stock solutions):

 Vildagliptin-
1. Crushed powder of drug obtained from tablet, equivalent to 10mg of drug was accurately
weighed and added to 100 ml distilled water.
2. The solution was stirred for about 10-15 minutes. The solution was filtered using Whatmann
Filter paper to remove the excipients and the resultant solution was used for UV analysis.

 Linagliptin-
1. Crushed powder of drug obtained from tablet, equivalent to 10 mg of drug was accurately
weighed and added to 100 ml ethanol.
2. The solution was stirred for about 10-15 minutes.

3. The solution was filtered using Whatmann filter paper to remove the excipients and
the resultant solution was used for UV analysis.

3. Determination of Detection Wavelength:

The solutions were scanned in the UV range 200-400 nm. From the spectra, 255.20 nm
wavelength was selected as detection wavelength for Vildagliptin and 236.80 was
selected as detection wavelength for Linagliptin.

4. Determination of Linearity
Linearity of an analytical method is its ability to elicit test results that are directly or by a
well-defined mathematical transformation, proportional to concentration of analyte in
samples within a given range.
 The linearity of an analytical method is determined by mathematical treatment of test results
obtained by analysis of samples with analyte concentrations across the claimed range. Area is plotted
graphically as a function of analyte concentration.

Preparation of Linearity solutions:

From the above prepared 100ppm solutions of Vildagliptin and Linagliptin, dilutions having
five different concentration range of analyte are prepared.

The dilution preparation for Vildagliptin is shown in the table.

Linearity Levels ml added Diluted to (ml) Concentration
(Stock Solution) (ppm)
Lin 1 1.0 10 10
Lin 2 2.0 10 20
Lin 3 3.0 10 30
Lin 4 4.0 10 40
Lin 5 5.0 10 50
Lin 6 6.0 10 60
The dilution preparation for Linagliptin is shown in the table.

Linearity Levels ml added Diluted to (ml) Concentration

(Stock Solution) (ppm)

Lin 1 0.5 10 5
Lin 2 1.0 10 10
Lin 3 1.5 10 15
Lin 4 2.0 10 20
Lin 5 2.5 10 25
Lin 6 3.0 10 30
5. Forced Degradation Study by UV Spectrometer
•Preparation of 1000ppm stock solution of Vildagliptin:
1. 20 Tablets of Gliptagreat each of 50mg equivalent to 1000
mg of vildagliptin were taken and crushed to form powder.
2. This powder was added to 1000ml distilled water and
stirred for about 10-15 minutes to facilitate proper mixing.
3. The solution was filtered to remove the excipients. The
resultant solution was used for further process.
• Preparation of 1000ppm stock solution of
Linagliptin:
1. 20 tablets of Linapil each of 5mg equivalent to 100 mg of
Linagliptin were taken and crushed to form powder.
2. This powder was added to 100ml ethanol and stirred for
about 10-15 minutes to facilitate proper mixing.
3. The solution was filtered to remove the excipients.

4. The resultant solution was used for further process.

•Preparation of 0.1N Sodium Hydroxide (NaOH) solution:

1. 0.84g of sodium hydroxide pellets were accurately weighed and transferred to a beaker containing 200 ml of

distilled water.

2. The solution was stirred to facilitate proper mixing.

•Preparation of 0.1N Hydrochloric acid (HCL):

1. 2.1ml of concentrated HCL was pipetted and transferred to 250 ml volumetric flask.

2. Volume was made to 250ml using distilled water.

•Preparation of 3% Hydrogen Peroxide (H2O2) solution:

1. 75ml of 6% H2O2 solution was transferred to a beaker followed by addition of 75ml distilled water.
 Acid Degradation:
•Method: 10ml stock solution of Vildagliptin and Linagliptin
were pipetted out and transferred to 100ml volumetric flask
and volume was made up to mark of 100 ml using 0.1N HCL.
Then the solutions were subjected to forced degradation at
room temperature for about 48 hours. After 48 hours, 1ml
solution was pipetted out from each flask and transferred to
10ml volumetric flask and volume was made up to 10ml mark
using water for Vildagliptin and ethanol for Linagliptin. The
absorbance of these solutions was recorded using UV
Spectrophotometer at a wavelength of 255.20nm for
Vildagliptin and 236.80nm for Linagliptin.
 Alkali Degradation:
•Method: 10ml stock solution of Vildagliptin and Linagliptin were pipetted out and
transferred to 100ml volumetric flask and volume was made up to mark of 100 ml using
0.1N NaOH. Then the solutions were subjected to forced degradation at room
temperature for about 48 hours. After 48 hours, 1ml solution was pipetted out from each
flask and transferred to 10ml volumetric flask and volume was made up to 10ml mark
using water for Vildagliptin and ethanol for Linagliptin. The absorbance of these solutions
was recorded using UV Spectrophotometer at a wavelength of 255.20nm for Vildagliptin
and 236.80nm for Linagliptin.
 Oxidative Degradation:
•Method: 10ml stock solution of Vildagliptin and Linagliptin
were pipetted out and transferred to 100ml volumetric flask
and volume was made up to mark of 100 ml using 3% H2O2.
Then the solutions were subjected to forced degradation at
room temperature for about 48 hours. After 48 hours, 1ml
solution was pipetted out from each flask and transferred to
10ml volumetric flask and volume was made up to 10ml mark
using water for Vildagliptin and ethanol for Linagliptin. The
absorbance of these solutions was recorded using UV
Spectrophotometer at a wavelength of 255.20nm for
Vildagliptin and 236.80nm for Linagliptin.
Results
 VILDAGLIPTIN:

1. The solution was scanned in the UV range 200-400 nm. From the spectra of drug, 255.20 nm
wavelength was selected as detection wavelength by using water as a solvent.
2. The following linearity graph was obtained when dilutions of 10, 20, 30, 40, 50, 60 ppm were
scanned at wavelength 255.20nm. Abs
0.45

0.4
f(x) = 0.00317714285714286 x + 0.202466666666667
0.35 R² = 0.999749902979604

0.3

0.25

0.2

0.15

0.1

0.05

0
0 10 20 30 40 50 60 70
Linearity data for Vildagliptin

Sr.no Concentration (ppm) Absorbance

1 10 0.235
2 20 0.265
3 30 0.298
4 40 0.334
5 50 0.360
6 60 0.394
Force Degradation study data:

Reagent Concentration Wavelength Absorbance % Recovery %

(ppm) (nm) Degradation

0.1N NaOH 10 255.20 0.084 48.08 51.92

0.082
0.173
0.1N HCL 10 255.20 0.079 35.74 64.26
0.090
0.084
3% H2O2 10 255.20 0.076 31.34 68.66
0.071
0.078
• Linagliptin
1. The solution was scanned in the range of 200-400 nm. From, the spectra of the drug, 236.80 nm
wavelength was selected as detection wavelength by using ethanol as a solvent.

2. The following linearity graph was obtained when dilutions of 5, 10, 15, 20, 25, 30 ppm were
scanned at 236.80 nm wavelength.

Abs

0.9
f(x) = 0.0196342857142857 x + 0.2714
0.8 R² = 0.998321993854188
0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
0 5 10 15 20 25 30 35
Linearity data for Linagliptin:

Sr.no. Concentration (ppm) Absorbance

1. 5 0.380
2. 10 0.463
3. 15 0.559
4. 20 0.654
5. 25 0.760
6. 30 0.869
 Force Degradation study data:

Reagent Concentration Wavelength Absorbance % Recovery %

(ppm) (nm) Degradation

0.1N NaOH 10 236.80 0.646 150.81 50.81

0.674
0.623
0.1N HCL 10 236.80 0.257 62.70 37.3
0.366
0.184
3% H2O2 10 236.80 1.354 310.25 210.25
1.308
1.328
Conclusion
A sensitive and selective UV-Spectroscopic method has been developed for the
analysis of Vildagliptin and Linagliptin. Stress testing studies are conducted to challenge the
specificity of stability-indicating and impurity-monitoring methods as a part of the validation
protocol. The choices of stress conditions were consistent with product decomposition under
normal manufacturing, storage, and use conditions. In this experiment, the purposefully
degraded Vildagliptin and Linagliptin were used to challenge the ability of the stability
indicating methods and the degradation was found in case of vildagliptin and when linagliptin
was treated with 0.1N HCL and degradation was unpredictable when Linagliptin was treated
with 0.1N NaOH and 3% H2O2.
According to ICH guidelines, a common goal is to introduce a significant degradation
typically between 10% - 30% to evaluate the method’s ability to detect and quantify
degradation products. However, in our case of Vildagliptin and Linagliptin degradation was
found to be beyond limit in the range of 35% - 70%. Also, in case of Linagliptin when forcedly
degraded under oxidative and basic conditions, degradation was unpredictable in nature.
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Thank You!