1.

INTRODUCTION

Analytical chemistry studies and uses instruments and methods to separate,

[1]
identify, and quantify matter. In practice, separation, identification or
quantification may constitute the entire analysis or be combined with another
method. Separation isolates analytes.

Qualitative analysis identifies analytes, while quantitative analysis

determines the numerical amount or concentration. Analytical chemistry
consists of classical, wet chemical methods and modern, instrumental
methods.[2]

According to need of analysis analytical chemistry can be classified as

following types

Qualitative
Analytical
chemistry
Quantitative

Fig no:1 classification of analytical chemistry

Qualitative - analysis which reveals determine the chemical identification

of the constituent species which may be of organic or inorganic in a sample.

Quantitative - analysis use to determine or establish how much of a

constituent species may be organic or inorganic is present in a sample.

Chemical method of analysis involves measurement of the mass or the

volume of the substances of reacting solution,

E.g. Gravimetric method, volumetric method, etc.

Instrumental methods which depends upon the measurement of electrical,

optical thermal and other properties and those based upon determination of
the extent to which radiation is absorbed or upon assessment of the intensity
of emitted radiation,
it requires the use of a suitable instrument e.g. spectrophotometer, HPLC,
oven, NMR etc.

1.1 Techniques of instrumental analysis

 Separation techniques:
These are the chromatographic method i.e. TLC, GC or HPLC which may
applicable for an analysis depends on various parameter like solubility or
volatility of the sample, separation efficiency, concentration of analyte, limit
of detection, analysis cost etc.

 Spectrometric techniques:

It includes UV, IR, NMR, MASS etc. spectrometric techniques are usually
used in the analysis of the drug of interest alone in the matrix of excipient,
degradation product, impurities, additives, etc. it also includes plasma,
atomic, X- ray flame, absorption or emission spectrometry etc.
 Electro-analytical techniques:
Electro- analytical method of analysis deals with electrical signal to the
sample and monitor the electrical property of the sample e.g. Aerometry,
polarography, electrogravimetry, conductometry, potentiometric, etc.
 Thermos- analytical techniques:
This analytical technique includes interaction of heat with the material. g
DSC, DTA, thermos Gravimetric technique, etc.

1.2 Spectroscopy-Spectroscopy is the measurement and interpretation of

Electro Magnetic Radiation [EMR] absorbed and emitted when the molecules
or atoms or ions of a sample move from one energy states to another energy
states.

1.2.1 UV – visible spectroscopy method

UV spectroscopy is the absorption or reflectance spectroscopy of the ultraviolet and
adjacent visible regions of the electromagnetic spectrum. It is also known as UV-
visible spectrophotometry (UV-Vis or UV/Vis). Because of its low cost and ease of
implementation, this methodology is widely used in a wide range of applied and
fundamental applications. The only requirement is that the sample absorb in the UV
Vis range, indicating that it is a chromophore. Absorption spectroscopy supplements
fluorescence spectroscopy. Aside from the wavelength, the parameters of interest are
absorbance (A), transmittance (%T), and reflectance (%R), as well as their
variations over time. [3]

Principle- The UV-Visible Principle The absorption of ultraviolet or visible

light by chemical compounds produces distinct spectra, which is the basis for
spectroscopy. The interaction of light and matter is the foundation of
spectroscopy. When matter absorbs light, it experiences excitation and de-
excitation, which results in the formation of a spectrum. [4]

When an electromagnetic wave strikes a material, phenomena such as

transmission, absorption, reflection, and scattering can occur, and the
observed spectrum depicts the interaction of wavelengths with discrete-
dimensional objects such as atoms, molecules, and macromolecules.
Absorption occurs when the frequency of incoming light equals the energy
difference between the ground and excited states of a molecule. An electronic
transition describes the excitation of an electron from its ground state to its
excited state.

Electrons will be promoted from their ground state orbital to a higher energy,
excited state orbital by the energy from the light. or anti-bonding orbital.
Potentially, three types of ground state orbitals may be involved.

1. σ (bonding) molecular

2. π (bonding) molecular orbital

3. n (bonding) atomic orbital [5]

1.2.2 Ultraviolet Absorption Spectrophotometry

Spectrophotometry is generally preferred especially by small-scale industries
as the cost of the equipment is less and the maintenance problems are
minimal. The method of analysis is based on measuring the absorption of a
monochromatic light by colourless compounds in the near ultraviolet path of
the spectrum (200- 400nm). The fundamental principle of operation of
spectrophotometer covering UV region consists in that light of definite
interval of wavelength passes through
a cell with solvent and falls on to the photoelectric cell that transforms the radiant
energy into electrical energy measured by a galvanometer. Ultraviolet visible
spectroscopy is used to obtain the absorbance spectra of a compound in solution or as
a solid.

1.2.3 Beer’s law and Lamberts Law

The intensity of beam of monochromatic light decreases rapidly with the

increase in concentration of the absorbing substance or it state the when the
light beam is passed through the solution of absorbing substance, the rate of
decrease of intensity of radiation with thickness of absorbing solution is
proportional to intensity of incident as well as the concentration of solution or
Beer’s law states that concentration and absorbance is directly proportional to
each other. [6-8]

Lambert’s Law
When the beam of light from radiation source is allowed to pass through a
transparent medium, the rate of decrease of intensity with the thickness of
medium is directly proportional to the intensity of light or the rate of decrease
of intensity of the
Monochromatic light with the thickness of medium is directly proportional to
the intensity of incident light. [9]
The Beer and Lambert law are normally combined in relation- A= -log10

P/P0 = abc

Where,
A = absorbance / optical density P = radiant power / intensity
a = absorptivity / extinction coefficient
b = length of the beam in the absorbing medium
c = concentration of the absorbing species [10]

1.2.4 UV-Visible spectra:

1. Single beam spectrometer - All of these instruments have a light source
(usually a deuterium or tungsten lamp), a sample holder and a detector, but
some have a filter for selecting one wavelength at a time. The single beam
 instrument has a filter or a monochromatic between the source and the
sample to analyze one wavelength at a time.
2. Double beam spectrometer-The double beam instrument has a single source
and a Monochromatic and then there is a splitter and a series of mirrors to get
the beam to a reference sample and the sample to be analysed, this allows for
more accurate Monochromatic between the sample and the source; instead, it
has a diode array detector that allows the instrument to simultaneously detect
the absorbance at all wavelengths. The simultaneous instrument is usually
much faster and more efficient.

1.2.5 Instrumentation [ 11-12]

 Sources (UV and visible)

 Monochromatic
 Sample containers (Cuvette)
 Detector
 Amplifier and recorder

Fig no 2 :Instrumentation of UV spectrophotometer

 Sources-A continuous source, or one that produces radiation at a variety of
wavelengths, is necessary for UV-Vis Spectroscopy. Assorted UV radiation
sources include the following:
 Hydrogen lamp: Hydrogen lamps are reliable, steady, and continuously emit
radiation between 160 and 380 nm. It consists of hydrogen gas at high
pressure, which causes an electrical discharge. The excited hydrogen
molecules produce radiation
 Deuterium lamp: A gas discharge lamp called a deuterium lamp is
frequently employed as a UV source. It emits radiation in the 160–450nm
range. It costs more than a hydrogen lamp.
 Tungsten lamp: The most typical light source utilized in spectrophotometers
is the tungsten lamp. With a wavelength range of roughly 330 to 900 nm, it
comprises of a tungsten filament encased in a glass envelope and is utilized
for the visible spectrum.
 Xenon discharge lamp: A xenon lamp is a discharge light source that
contains xenon gas inside a bulb. Radiation from xenon ranges from 250 to
600 nm.
 Monochromator: By filtering out undesirable wavelengths from the
radiation source light, a monochromator creates monochromatic light.
Through the entrance slit, multi-wavelength polychromatic light enters the
monochromator. Following collimation, the beam is directed at an angle
toward the dispersion component. The grating or prism separates the beam's
wavelengths into their individual components. Only radiation of a specific
wavelength exits the monochromatic through the exit slit when the dispersing
element or the exit slit are moved.
o Types of monochromators:
 Prism monochromatic
 Grating monochromatic
 Sample Containers (Cuvette): Cuvettes are sample containers that are
transparent to all wavelengths of light flowing through them and are used to
hold samples for spectroscopic measurements. The cuvette is composed of
 quartz, is square in shape, has a 1 cm route length, and may be utilized for
wavelengths between 190 and 200 nm.
 Detectors: Light energy is converted by detectors into electrical impulses
that are read out by readout devices. The transmitted radiation strikes the
detector, determining the amount of radiation absorbed by the sample. The
absorption spectrophotometer's apparatus uses the following types of
detectors.
o Types of Detectors:
 Barrier layer cell/Photovoltaic cell
 Phototubes/ Photo emissive tube
 Photomultiplier tube

Application of UV visible Spectroscopy

1. Detection of impurities
2. Structural elucidation of organic compounds
3. Quantitative analysis
4. Qualitative analysis
5. Chemical analysis
6. Quantitative analysis of pharmaceutical substance
7. Dissociation constant of acids and bases
8. Molecular weight determination
9. As HPLC detector
10. Deviations from the Beer-Lambert law. [13-16]

1.3 Chromatography

1.3.1 Introduction - The term "chromatography" finds its roots in two Greek
words, "Chroma" and "graphein," both meaning "to write. “Chromatography
is a laboratory technique for the separation of a mixture. The mixture is
dissolved in a fluid called the mobile phase, which carries it through a
structure holding another material called the stationary phase.

1.3.2 Principle- Chromatography is based on the principle where molecules

in mixture applied onto the surface or into the solid, and fluid stationary
phase
 (stable phase) is separating from each other while moving with the aid of a
mobile phase. The factors effective on this separation process include
molecular characteristics related to adsorption (liquid-solid), partition (liquid
solid), and affinity or differences among their molecular weights. Because of
these differences, some components of the mixture stay longer in the
stationary phase, and they move slowly in the chromatography system, while
others pass rapidly into mobile phase, and leave the system faster. [17]

Three components thus form the basis of the chromatography technique.

 Stationary phase: This phase always composed of a “solid” phase or “a

layer of a liquid adsorbed on the surface a solid support”.

 Mobile phase: This phase is always composed of “liquid” or a

“gaseous component”.

Separated molecules.

The type of interaction between the stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on
separation of molecules from each other.

1.3.3 Chromatography techniques

The different chromatographic techniques include:

 Thin-layer chromatography
 Gas chromatography
 High performance thin-layer chromatography
 High-pressure liquid chromatography (HPLC)
 Ultra-violet high performance liquid chromatography
 GC-MS (Gas chromatography- Mass spectroscopy)
 LC-MS) Liquid Chromatography-Mass Spectroscopy)
 Fig no 3: Classification of chromatography

1.3.4 High performance liquid chromatography

HPLC, standing for high-performance liquid chromatography, is a widely

used method for pharmaceutical analysis due to its simplicity and versatility.
It is a comprehensive process that involves the use of specific equipment
known as an HPLC system. HPLC serves various purposes:

1) Recognizing specific components or groups of compounds in a sample,

e.g., determining the presence and quantity of diazepam in a blood sample or
caffeine in a plant extract.

2) Identifying unknown components in a sample requires the use of

detection aids like mass spectrometers. Collecting eluent fractions with the
peak of interest for off-line characterization is often essential [12], [18]
1.3.5 Modes of separation of HPLC

 Normal Phase Chromatography

 Reversed Phase Chromatography
 Ion exchange Chromatography
 Size exclusion Chromatography
 Normal phase chromatography: Section HPLC- in this approach the
separation is based totally on polarity. The stationary phase is polar, broadly
speaking Silica is used and the non-polar segment used is hexane, chloroform
and diethyl ether. [19]
 Reversed Phase Chromatography: It is reverse to normal phase HPLC. The
mobile phase is polar and the stationary phase is non polar or hydrophobic.
The more is the non-polar nature the more it will be retained.
[20]

 Ion exchange Chromatography: The ironically charged surface of the

stationary phase is the opposite of the charge on the sample. Aqueous buffer
is utilised as the mobile phase and will regulate the pH and ionic strength.
[21]

 Size exclusion Chromatography: The substrate molecules will be added to

the column in a precisely regulated manner. The separation of constituents
will take place based on the variation in molecular sizes. [22]

1.3.6 Method development in HPLC

The goal of the HPLC methods is try to separate and quantify the main active
drug, any reaction impurities, all available synthetic inter- mediates and any
degradants. The number of drug 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.
Sample Information:
The knowledge of nature and type of sample play an important role in the
development of new HPLC method. It is necessary that analyst should have
information that whether sample is polar or non-polar in nature. Ultimately
this gives the idea of selection of stationary phase (Column packing material)
for proper chromatographic separation of compounds. Pure drugs, their
impurities and degradation products, excipients, finished dosage forms
should be collected and the method should be designed based on the best
resolution between the closely related compounds. The structures of
impurities, starting material, intermediates and degradation products are
compared with the structure of drug substances and arrive at the polarity
whether they are less polar or more polar than the compound of interest.
Selection of Detector:
The selection of detector is based on the presence or absence of
chromophores in the analyte. But, majority of pharmaceutical compounds
exhibit UV spectra in the range of 200-400 nm. The most common detectors
for HPLC system UV- Visible detector but PDA (Photodiode Array) detector
is now a day widely used for identification of drug impurities and
degradation products. The non- chromophore compounds can be analyzed by
using fluorescence, mass detectors etc. To confirm the peak purity, PDA
detector is employed in modern HPLC/UPLC system. It collects the spectra
at each point of the peak, starting from the peak elution to peak end and
compares the spectra of peaks collected at all points versus the peak apex and
thus confirms the homogeneity of the peak. Selection of Mobile Phase:
The mobile phase selection is one of the critical parameter as it encourages
the solute and the stationary phase interactions. An appropriate care must be
taken while selecting the mobile phase like use of strong acids, strong bases
and halide solutions should be avoided.
Selection of Solvents:
Another essential parameter for HPLC method development is solubility of
the compounds in which it is dissolved. The solvent should be chosen based
on the solubility of drug substances, their impurities and degradation
products. The
 solvent should be compatible with the mobile phase to get a better peak shape
of analyte. Consider the purity of solvents and only use HPLC grade solvents.
In normal phase HPLC systems, non-polar solvents such as hexane, diethyl
ether, dichloromethane, iso-propyl alcohol, iso-octane etc. are used whereas
reversed phase HPLC requires polar solvents such as water, acetonitrile
ethanol or methanol. The choice of mobile phase is governed by the physical
properties of the solvent. Factors which are considered to be essential for
selection of particular solvent are polarity, miscibility with other solvents,
chemical inertness, and toxicity. The polarity index gives an indication of the
ability of a solvent to elute a compound from the column.
Selection of Buffer pH and Type of Buffer:
Buffers are usually employed in the mobile phase to obtain consistent
chromatographic results. Buffers are employed to control the retention of
ionic analytes. When the analyte is in ionic form, it usually attains polar in
nature and spends shorter time on the stationary phase and elutes quickly. To
control the selectivity of the ionic analytes, the buffer pH plays a significant
role. In general, when buffer pH increases, the acidic analytes gets ionized
and become more polar in nature and conversely, when buffer pH decreases,
the basic analytes gets ionized. The pH of the mobile phase selected should
be at least 1.0 pH units from the analyt pKa value. This confirms that the
analytes are either as 100% ionized or 100% non-ionized and it helps in
controlling peak shape and the run to run reproducibility. It is always use
buffer in aqueous portion of the mobile phase and it increases the ruggedness
of the method.
1.3.7 Components of HPLC System:
1. Solvent reservoirs
2. Degasser
3. Pump
4. Column
5. Detector
6. Data recording [23]
1. Pump: Compared to gravity-flow columns, a pump propels the mobile
phase through the column at a significantly higher speed. Even when the
 mobile phase's composition changes, the pump is built to maintain a steady
flow rate and prevent pulsations.
2. Detector: As each separated component exits the column, the HPLC detector
measures the eluent and generates an electrical signal proportionate to its
concentration. The detectors with the greatest use: Fluorescence,
conductivity, evaporative light scattering, refractive index detectors are
examples of detectors. [24]
3. Column: It is necessary to explain the significance of the column because it
is a crucial part of HPLC. The most popular and often used system is an
HPLC column, which is made of packing made of silica.
4. Injector: The injector is generally utilised to inject liquid samples. There are
two different types of injectors: automated and manual.
5. Data recording: The detected signal is transformed to an electrical signal
when detection is complete, which is then amplified by an amplifier and
recorded as a chromatogram in data points. Then, use the programme as the
display format in accordance with the manual or software conversion
requirements

Fig no 4. Instrumentation of HPLC

1.3.8 Parameters Used in Chromatogram Characterization: System

suitability-
Selection a proper system suitability testing mixture is essential to check the
specification of a liquid chromatographic system. SST limits are acceptance
criteria that must be met prior to sample analysis. It is used to verify the
resolution, column efficiency, and repeatability of the analysis system to
ensure its adequacy for performing the intended application on the daily
basis. According to the latest USP and ICH guidelines, SST has must be
performed before and throughout all regulated assays. Primary SST
parameters are resolution, repeatability (RSD of peak response and retention
time), column efficiency (N), and tailing factor (TF). [25]

Efficiency

In column chromatography, a number of theoretical plate N (based on the

theoretical plate concept of distillation columns), is used to measure the
efficiency of column. It can be calculated by using the formula-

N=16(tR/W) 2 or N-5.54(tR/Wh/2) ² Where,

tR -the retention time of analyte.

W- the peak width measured in time units as the distance between the
intersections of the tangents to the peaks inflection point with the baseline.

Wh -Peak width at half height.

Resolution-

The resolution is used to check whether the critical separation is feasible

under the given condition. The very high plates numbers are giving by
column are capable of separating multi component mixtures, but it is their
resolving power, as measured by the resolution (Rs), that is of prime
importance. By convention, resolution is expressed as the ratio of the distance
between two peak maxima to the mean value of the peak width at the
baseline, equation-

Rs= 2 ∆tr/(W1+W2)

Where,
 ∆tr =Difference between retention times of two adjacent solute peaks. WI &

W2=Base widths of two adjacent peaks. [26]

Tailing factor-

This is also called as symmetry factor Tailing factor become important if the
peak tailing has the chances of affecting the methods performance. It was
developed by USP as a parameter to determine peak asymmetry.

It can be calculated using formula-

T0.05=W0.05/2 Wa

Where,

W0.05 = the width of peak at 5% height

Wa = Distance from the peak maximum to the leading edge of the peak

Capacity factor (K')

The capacity factor is a way of expressing the retention of the compound. It

can be given as:

K'=(Tr-T0) T0

Where,

Tr- The retention time of retained analyte.

TO -The time required for unrestrained non-excluded peak, [27]

1.3.9 Method validation

Validation parameters:

The method validation parameters as per ICH guideline are

 Accuracy
 Precision
 Repeatability
 Reproducibility
 Intermediate precision
 Specificity
 Limit of detection
 Limit of quantitation
 Linearity
 Range
 Ruggedness
 Robustness
 System suitability testing
 Accuracy- It is the closeness of mean tests results obtained by the method to
true concentration of analyte. It is also named as trueness. Accuracy is
determined by replicate analysis of samples containing known amounts of the
analyte. Most commonly used method for determination of accuracy is
recovery studies. The usual range is being 10% above or below the expected
range of claim.
The recovery was calculated using the formula,
% Recovery = (axb)-a hx100 Where,
a = Amount of drug present in sample
b = Amount of standard added to the sample Acceptance Criteria:
In assay method, mean recovery will be 100% 2% at each concentration
between the ranges of 80-120% of the target concentration.
In impurity method, mean recovery will be 0.1% absolute of the theoretical
concentration or 10% relative, whichever is greater for impurities between
the ranges of 0.1-2.5% (V/W), [28]
 Precision –When the procedure is applied repeatedly to multiple samplings
of single homogenous sample under prescribed conditions then precision, is a
closeness of individual measurements of the analyte. It is done at three levels
such as repeatability, intermediate precision, and reproducibility.
 Repeatability: It expresses precision under same operating conditions ie,
within the laboratory same analyst using same equipment over a short period
of time.
 Intermediate precision: It is the precision under different laboratory
conditions ic. varying only in different analyst, on different days, or using
different equipment's within the same laboratory.
 Reproducibility: It is the precision between different laboratories and is
often determined in method transfer experiments
Acceptance Criteria:
Percentage Relative deviation (RSD) NMT 1% (Instrument precision)
(%RSD) NMT-2% (Intra- assay precision) [28,29,30]
 Linearity: As per ICH definition "the ability to obtain test results which are
directly proportional to the concentration of an analyte within given range is
known as linearity of an analytical procedure" By using correlation
coefficient this can be tested. Using correlation coefficient is a benefit as it is
a relationship between concentration and response data. In this data is
analysed by linear least square regression co-efficient and b of the linear
equation.
Y=mx+c
By the above equation regression r value can be known. For the method to be
linear the r value should be close to 1.
Where Y is the measured output signal, x is the concentration of sample, m is
the slope, c is the intercept. [31]
Acceptance criteria:
Coefficient of correlation should be NLT 0.999
 Specificity/selectivity: A method is said to be specific when it produces
proper response only for a single analyte. It can be demonstrated by
performing Placebo/blank interference and forced degradation studies. If the
expected impurities or related substances are available, then they should be
analysed along with the analyte or sample to check the system suitability,
retention factor, tailing factor and resolution etc. In this peak purity studies
are done for specificity. [28,31]
 Limit of Detection (LOD): The limit of detection is the lowest
concentration of analyte in the sample which can be detected but not
quantified under given experimental conditions. The lowest concentration
which can be distinguished from the background noise with a certain degree
of confidence is defined as limit of detection. Prepare the blank solution as
per test method and inject six times into the chromatographic system.
Similarly prepare the linearity solution staring from lowest possible
concentration of analyte to 150% (or as per protocol) of target concentration
and establish the linearity curve.
The detection limit may be expressed as:
LOD- 3.3 σ/S
Where, σ = the standard deviation of the response S = the slope of the
calibration curve.
The slope shall be estimated from the calibration curve of the analyte.
 Lower Limit of Quantification (LOQ): It is also the lowest concentration
of analyte in the sample but quantitatively determined with suitable accuracy
and precision. In calibration curve it is the lowest concentration point. It is
determined by accuracy by the presence of background signal and by
precision i.e., reproducibility of analyte in the method.
LOQ = 10 σ/ S
Where, σ= the standard deviation of the response S= the slope of the
calibration curve.
Acceptance Criteria:
In Pharmaceutical application, the LOQ is typically set at minimum 0.05%
for active pharmaceutical ingredients.
LOQ defined as the lowest concentration providing an RSD of 5%.
LOQ should be at least 10% of the minimum effective concentration for
clinical applications. [32]
 Range: The range of an analytical procedure is the interval between the
upper and lower concentration (amounts) of analyte in the sample (including
these concentrations) for which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and linearity. The range
 of a bioanalytical assay is the concentration interval over which an analyte
can be measured with acceptable precision and accuracy.
 Robustness: It is the measure of its capacity to remain unaffected by small,
but deliberate variations in method parameters and provides an indication of
its reliability during normal usage.
 Ruggedness: Ruggedness according to the USP is "the degree of
reproducibility of test results obtained by the analysis of the same samples
under a variety of normal test conditions, such as different labs, different
analysts, and different lots of reagents. The following are the typical method
parameters need to test during method validation:
 Analyst-to-analyst variability.
 Column-to-column variability.
 On different days.
 In different laboratories [33]
 System suitability: System suitability testing is an integral part of analytical
procedures. System suitability test parameters to be established for a
particular procedure depend on the type of procedure being validated. The
simplest form of system suitability test involves a comparison of the
chromatogram trace with a standard trace. This allows a comparison of the
peak shape, peak width, and baseline resolution. These are the parameters
that can be calculated experimentally to provide a quantitative system
suitability test report.
 Number of theoretical plates (efficiency)
 HETP
 Capacity factor,
 Peak asymmetry factor
 Resolution,
 Tailing factor
1.4 Stability Indicating Assay Methods [34,35]

Stability-indicating methods according to guideline were defined as the

'quantitative analytical methods that are based on the characteristic structural,
chemical or biological properties of each active ingredient of a drug product
and that will distinguish each active ingredient from its degradation products
so that the active ingredient content can be accurately measured. The
stability- indicating assay is a method that is employed for the analysis of
stability samples in pharmaceutical industry.

With the advent of International Conference on Harmonization (ICH)

guidelines, the requirement of establishment of stability-indicating assay
method (SIAM) has become more clearly mandated. The guidelines
explicitly require conduct of forced decomposition studies under a variety of
conditions, like pH, light, oxidation, dry heat, etc. and separation of drug
from degradation products. It is also mentioned that forced decomposition
studies (stress testing) at temperatures in10°C increments above the
accelerated [36,37]

Temperature extremes of pH and under oxidative and photolytic conditions

should be carried out on the drug substance so as to establish the inherent
stability characteristics and degradation pathways to support the suitability of
the proposed analytical procedure.

Force degradation study –

Typical stress tests include four main degradation mechanisms: heat,

hydrolytic, oxidative, and photolytic degradation. Selecting suitable reagents
such as the concentration of acid, base, or acidizing agent and varying the
conditions (e.g., temperature) and length of exposure can achieve the
preferred level of degradation. Overstressing a sample may lead to the
formation of secondary degradants that would not be seen in formal shelf-life
stability studies and under- stressing may not serve the purpose of stress
testing. Therefore, it is necessary to control the degradation to a desired level.
A generic approach for stress testing has been proposed to achieve purposeful
degradation that is predictive of long- term and accelerated storage
conditions.
 The generally recommended degradation varies between 5-20% degradation
This range covers the generally permissible 10% degradation for small
molecule pharmaceutical drug products, for which the stability limit is 90%-
110% of the label claim. Although there are references in the literature that
mention a wider recommended range (e.g. 10-30%), the more extreme stress
conditions often provide data that are confounded with secondary
degradation products.

1.4.1 Hydrolytic condition

Hydrolysis is one of the most common degradation chemical reactions over

wide range of ph. Hydrolysis is a solvolytic process in which drug reacts
with water to yield breakdown products of different chemical compositions.
Water either as a solvent or as moisture in the air comes in contact with
pharmaceutical dosage forms is responsible for degradation most of the
drugs. For example, aspirin combines with water and hydrolysed to salicylic
acid and acetic acid. Hydrolytic study under acidic and basic condition
involves canalization of ionisable functional groups present in the molecule.
HCI and NaOH are employed for generating acidic and basic stress samples,
respectively, the hydrolytic degradation of a new drug in acidic and alkaline
condition can be studied by refluxing the drug in 0.1 N HCI / 0.1 N NaOH.
If reasonable degradation is seen, testing can be stopped at this point.
However, in case no degradation is seen under these conditions the drug
should be refluxed in acid/alkali of higher strength and for longer duration
of time. Alternatively, if total degradation is seen after subjecting the drugs
to initial condition, acid/alkali strength can be decreased with decrease in
reaction temperature. In general temperature and pH are the major
determinant in stability of the drug prone to hydrolytic decomposition.
Hydrolysis of most of the drugs is dependent upon the relative concentration
of hydronium and hydroxyl ions. Hence pH at which each drug is optimally
stable can be determined.

1.4.2 Oxidation conditions

Hydrogen peroxide is widely used for oxidation of drug substances in forced

degradation studies but other oxidizing agents such as metal ions, oxygen,
and
 radical initiators (e.g., azobisisobutyronitrile, AIBN) can also be used.
Selection of an oxidizing agent, its concentration, and conditions depends on
the drug substance. It is reported that subjecting the solutions to 0.1-3%
hydrogen per oxide at neutral pH and room temperature for seven days or up
to a maximum 20% degradation could potentially generate relevant
degradation products. The mechanism of oxidative degradation of drug
substance involves an electron transfer mechanism to form reactive anions
and cations. Amines, sulphides and phenols are susceptible to electron
transfer oxidation to give N-oxides, hydroxylamine, sulphates and sulphide
Diol The functional group with labile hydrogen like benzylic carbon, allylic
carbon, and tertiary carbon or a positions with respect to hetro atom is
susceptible to oxidation to form hydro peroxides, hydroxide or ketone.

1.4.3 Photo degradation

According to ICH QIB guideline for photo degradation, samples should be

exposed to light providing an overall illumination of not less than 12 million
lux hours and an integrated near ultraviolet energy of not less than 200 watt
hours/square meter with spectral distribution of 320-400nm to allow direct
comparisons to be made between the drug substance and drug product.
Samples may be exposed side by-side with a validated chemical
actinometrical system to ensure the specified light exposure is obtained, or
for the appropriate duration of time when conditions have been monitored
using calibrated radiometers/lux meters. The photolytic degradation can
occur through no oxidative or oxidative photolytic reaction. The no
oxidative photolytic reaction includes isomerization, dimerization,
cyclization, rearrangements. decarboxylation and haemolytic cleavage of X-
C hetero bonds, N-alkyl bond (DE alkylation and deamination), S02-C
bonds ete, and while oxidative photolytic reaction occurs through either
singlet oxygen (1O2) or triplet oxygen (3O2) mechanism. The singlet
oxygen reacts with the unsaturated bonds, such as alkenes, dienes,
polynuclear aromatic hydrocarbon to form photo oxidative degradation
products whereas triplet oxygen react with free radical of the drug
molecule, which than react with a
 triplet oxygen molecule to form peroxide. Hence, light can also act as a
catalyst to oxidation reactions.

1.4.4 Thermal condition

In general, rate of a reaction increase with increase in temperature. Hence,

the drugs are susceptible to degradation at higher temperature. Many APIs
are sensitive to heat or tropical temperatures. For example, vitamins,
peptides, etc. Thermal degradation involves different reactions like
pyrolysis, hydrolysis, decarboxylation, isomerization, rearrangement and
polymerization. Effect of temperature on thermal degradation of a substance
is studied through Arrhenius equation: K=Ae-Ea./RT Where k is specific
reaction rate. A is frequency factor, Ea. is energy of activation, R is gas
constant (1.987 Cal/ deg mole) and T is absolute temperature. Thermal
degradation study is carried out at 40°C to 80°C. The most widely accepted
temperature is 70°C at low and high humidity for 1- 2 months. High
temperature (>80°C) may not produce predictive degradation pathway. The
use of high-temperatures in predictive degradation studies assumes that the
drug molecule will follow the same pathway of decomposition at all
temperatures. This assumption may not hold true for all drug molecules, and
therefore great care must be taken in using the extreme temperatures easily
accessible in a sealed-vessel microwave experiment for predictive
degradation studies.
2. LITERATURE OF REVIEW

Literature review reveals that there are very few methods for the
determination of metformin HCl and sitagliptin phosphate individually in pure
drug. Details of few methods are reported are as follows:

[38]
2.1 Aqeela Raza et.al (2022) The Validation of a rapid and economical
RP- HPLC method for simultaneous determination of metformin
hydrochloride and sitagliptin phosphate monohydrate: Greenness
evaluation using AGREE score. In RP-HPLC method, the analytic was
resolved by using a gradient system, acidified water and methanol 60:40
(v/v) was used as mobile phase, at a flow rate of 1mL/min, on HPLC
system containing Shimadzou® C18 column (250mm × 4.6mm, 5μm). The
detection was carried out at 260nm at 25oC. The retention time was found
to be for MET and SIT were 1.96 and
3.70 min, the developed method was time saving, economical, rapid,
robust, rugged, precise, accurate and found to be applicable for
simultaneous determination of MET and SIT in commercial tablets.
[39]
2.2 Balamurugan Krishnan et.al (2020) Quality by Design based
Development and Validation of RP-HPLC Method for Simultaneous
Estimation of Sitagliptin and Metformin in Bulk and Pharmaceutical
Dosage Forms. The chromatographic separation for Monolithic C18
segment (100×4.6 mm id, 5µm molecule size) and PDA-UV- detection at
210nm.The mobile phase used methanol: 40- 50% v/v, pH: 3.5-4.5 and
flow rate:0.3- 0.5ml/min. metformin and sitagliptin eluting with retention
times of 3.3 and
4.4 min respectively. determined the assay was specific, accurate and
linear, precise and robust. this RP-HPLC method can be used as a routine
quality control analysis of gliptin derivative like sitagliptin in combination
with metformin.
2.3 P. B. N. Prasad et.al (2014) [40] have developed and Validated of a Method
for Simultaneous Determination of Metformin Hydrochloride and
Sitagliptin Phosphate in a Formulation by RP-HPLC. The mobile phase
used methanol: potassium di-hydrogen phosphate buffer at a ratio of 70:30
v/v. The chromatographic method was standardized using a Hibar-
240,
 Lichrosphere-100 C18 ODS (250 × 4.6 mm, 5 µm) column with UV
detection at wavelength of 266 nm.
[41]
2.4 Kavitha D. S. K. Sahoo et.al (2017) has Developed And Validated of
RP- HPLC Method For Determination Of Metformin And Sitagliptin In
Bulk And Pharmaceutical Dosage Form. The chromatographic separation
for Intersil-BDS C18 column (250 × 4.6 mm, 5 μm particle size). The
wavelength detector was set at 258 nm. The mobile phase used Water:
Methanol (60:40).
[42]
2.5 Srivani Mallepelli et.al (2017) has to develop a simple, rapid and
specific RP-HPLC method for the estimation of metformin and sitagliptin
in bulk and combined pharmaceutical dosage forms. The chromatographic
separation Luna C18 250mmx4.6mm, 5µ particle size, Elution mode was
Isocratic, Mobile phase: Buffer: ACN (50: 50) (v/v), Flow rate: 1.0 mL
/min, Detection wavelength 285 nm, to validate the proposed methods in
accordance with the analytical parameters mentioned in the ICH
guidelines, such as system suitability, accuracy, precision, specificity,
linearity, robustness.
2.6 Karam Qassas et.al (2015) [43] A simple, rapid and precise reversed-phase
High performance liquid chromatographic (RP-HPLC) stability indicating
method was developed and validated for the determination of Sitagliptin in
the bulk and tablets. Chromatographic separation of sitagliptin was
achieved using a reverse phase C18 column (250 mm × 4.6 mm, 5.0 µm)
with isocratic flow using a mobile phase consisting of methanol and
phosphate potassium buffer pH 6.8 (60:40, v/v). The flow rate was
1mL/min and the eluents were monitored at260 nm. The method was
successfully validated in compliance to ICH guidelines acceptance criteria
in terms of linearity, accuracy, precision, limit of detection, limit of
quantification and robustness where all validation parameters were within
the acceptance range.
2.7 Hemraj Sharma et.al (2019) [44] To develop and validate a rapid, specific,
accurate and precise Reverse phase High performance liquid
chromatographic (RP-HPLC) method for the simultaneous determination
of Metformin and Sitagliptin in pharmaceutical dosage forms and
its
 applications to dissolution study. The chromatographic separation was
carried out on a C8 (250mm X 4.6 mm i.e., 5μm) column with a mobile
phase of 40 Acetonitrile: 60 Phosphate Buffer (pH 6.8), using UV detector
at 257 nm at 1ml min-1 flow rate. The retention time for Metformin was
2.11 minutes and 5.30 minutes for Sitagliptin. The Linearity for Metformin
was found to be 10-80 µg ml-1 with R2 value of 0.9998 and for Sitagliptin
1-8 µg ml-1 with R2 value of 0.9976.
[45]
2.8 Gaddala Deepthi et.al (2019) A simple, rapid, precise, accurate and
sensitive reverse phase liquid chromatographic method has been developed
for the determination of Sitagliptin in bulk and pharmaceutical dosage
form dosage form. The chromatographic method was standardized using
Develosil ODS HG-5 RP C18, 5µm, 15cm x 4.6mm i.d. column with UV
detection at 255 nm and (0.05M) Phosphate Buffer: Acetonitrile with
30:70 (pH-2.8) ratio at a flow rate of 1.0 ml/ min. The proposed method
was successfully applied to the determination of Sitagliptin in bulk and
pharmaceutical dosage form.
[46]
2.9 Sai Lakshmi.E et.al (2017) Development and Validation of RP-HPLC
Method for the Estimation of Sitagliptin Phosphate in Tablet Dosage Form.
The chromatographic separation for Sitagliptin was achieved with mobile
phase containing methanol, Thermoscientific C18 column, (250x4.6
particle size of 5µ) at room temperature and UV detection at 248 nm. The
compounds were eluted in the isocratic mode at a flow rate of 1ml/min.
The retention time of Sitagliptin was 1.91min.
[47]
2.10 Imran A Sheikh et.al (2017) Application of Validated HPLC
Method for Degradation Study of Sitagliptin and Metformin HCl UV
DetectorC18 column (150 mm × 4.6 mm). A mixture of Potassium
Phosphate buffer pH-
3.2 with orthophosphoric acid and acetonitrile was used as mobile phase in
this method with flow rate 0.7 ml/min (UV detection at 203 nm) and the
method was validated as per ICH guidelines. Forced degradation studies
were performed by exposing the drug Sitagliptin and Metformin HCL to
acidic, alkaline, oxidation and thermal stress degradations.
 [48]
2.11 P. Ramalingam et.al (2014) A new stability-indicating high-
performance liquid chromatographic method for simultaneous analysis of
sitagliptin and simvastatin in pharmaceutical dosage form was developed
and validated. The mobile phase consisted of methanol and water
(70:30, v/v) with 0.2 % of n-heptane sulfonic acid adjusted to pH 3.0
with Orthophosphoric acid was used. Retentions of sitagliptin and
simvastatin were 4.3 min and 30.4 min, respectively with a flow rate of 1
ml/min on C8 (Qualisil BDS, 250×4.6 mm, 5 μ). Eluents were detected at
253 nm using photodiode diode array detector. Both drugs were subjected
to a variety of stress conditions such as acidic, basic, oxidation, photolytic,
neutral and thermal stress in order to achieve adequate degradation.
[49]
2.12 Sandhya Nune et.al (2017) Development of A Simple, Rapid and
Specific RP-HPLC Method for The Estimation of Metformin and
Sitagliptin in Bulk and Combined Pharmaceutical Dosage Forms. The
separation of chromatographic condition for Luna C18 250mmx4.6mm, 5µ
particle size, Mobile phase: Buffer: ACN (50: 50) (v/v), Flow rate: 1.0
mL /min, Detection wavelength 285 nm. To validate the proposed methods
in accordance with the analytical parameters mentioned in the ICH
guidelines, such as system suitability, accuracy, precision, specificity,
linearity, robustness.
2.13 Sudhir Adsul et.al (2018)[50] RP- HPLC method have been
Development and Validation for Simultaneous Estimation for Metformin
and Sitagliptin in Bulk and Tablet Formulation was developed using Grace
C18 column (250nm x 4.6ID, Particle size: 5 Micron) as stationary phase
and methanol: HPLC grade water (80:20%v/v, pH3.0) as mobile phase was
maintained at a flow rate of 0.8ml/min, the retention time of Metformin
and Sitagliptin were found to be 6.19 min and 7.42 min and detection was
carried out at 254nm.
3. Drug profile
3.1 Drug Name: Sitagliptin phosphate monohydrate
Name of Sitagliptin phosphate monohydrate
Drug
F
Chemical F
O
structure
F H H
F N
N
N
N
HO
OH
NH2 O
F P
F
HO
O

Chemical (R)-3-amino-1-(3-(trifluoromethyl)-5,6-
name dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-
yl)-4-(2,4,5-trifluorophenyl)butan-1-one
Phosphate Monohydrate.
Melting point 206.370C

Chemical C16H20F6N5O6P
formula
Molecular 523.32 g/mol
Weight
Category antidiabetic drug

Solubility soluble in water and N, N-dimethyl form

amide, slightly soluble in methanol, soluble in
ethanol, acetone and acetonitrile and insoluble
in isopropanol and isopropyl acetate.

Appearance White to off powder

Pka value 8.78

Log p 1.26
Half life 12.4 hours,Other studies have reported a half-
life of approximately 11 hours
Bioavailabilit 87%
Y
Dose 100mg
Description –Sitagliptin Phosphate is the phosphate salt form of sitagliptin,
an orally available, competitive, beta-amino acid-derived inhibitor of
dipeptidyl peptidase 4 (DDP-4) with hypoglycaemic activity. Sitagliptin may
cause an increased risk in the development of pancreatitis.

Mode of action – Sitagliptin and DPP-4 inhibitors in general address a novel

multimodal principle of action in type 2 diabetes. By preserving stimulated
circulating plasma levels of incretion hormones, insulin secretion is stimulated
under hyperglycaemic conditions and glucagon secretion is suppressed.

Pharmacokinetics-

 Absorption – Sitagliptin is 87% orally bioavailable and taking it with or

without food does not affect its pharmacokinetics. Sitagliptin reaches
maximum plasma concentration in 2 hours.
 Volume of distribution- 198L.
 Metabolism- sitagliptin is mostly not metabolized, with 79% of the dose
excreted in the urine as the unchanged parent compound. minor metabolic
pathways are mediated mainly by cytochrome p450(CYP)3A4 and to a
lesser extent by CYP2C8L label. After 18 hours, 81% of the dose has
remained unchanged, while 25 has been N-sulfated to the M1 metabolite,
6% has been oxidative desaturase and cyclized to the M2 metabolite.
 Route of elimination- sitagliptin is well absorbed and about 80% is
excreted unchanged in the urine; the half-life is 8-14 hours.
 Renal clearance – renal clearance averages 350ml/min and is largely
uninfluenced by dose. The AUC is dose-dependent and is not affected by
food.

Toxicity – Animal studies in pregnancy have shown no adverse effects on the

mother or offspring at normal doses, however these results are not always
applicable to humans there is a voluntary fetal exposure registry label, animal
studies at 100 times the maximum recommended human dose resulted in an
increase in rib malformations.
3.2 Drug Name: Metformin HCl

Name Metformin Hydrochloride

of drug
Chemical CH3
Structure
H2N
N N
CH3

NH2 NH
. HCl

Chemical name 3-(diaminomethylidene)-

1,1dimethylguanidine; hydrochloride
Chemical C4H11N5•HCl
Formula
Molecular 165.625 g/mol
weight
Melting point 219.0 to 223.0 °C
Category anti-diabetic
Solubility Freely soluble in water; slightly soluble
in alcohol; practically insoluble in
acetone and in methylene chloride.

Bioavailability 40 to 60 %
Pka value 12.4
Appearance white to off-white crystalline compound
Half life Between 4.0 and 8.7hours.

Description – Metformin HCl decreases the amount of glucose released into

the bloodstream from the liver and increases the body use of the glucose.

Mechanism of action -Metformin is an antihyperglycemic agent, which

improves glucose tolerance in patients with type 2 diabetes, lowering both
basal and postprandial plasma glucose. Its pharmacologic mechanisms of
action are different from other classes of oral antihyperglycemic agents.
Metformin decreases hepatic glucose production, decreases intestinal
absorption of glucose, and improves insulin sensitivity by increasing
peripheral glucose uptake and utilization. Unlike sulfonylureas, metformin
does not produce
 hypoglycaemia in either patient with type 2 diabetes or normal subjects and
does not cause hyperinsulinemia. With metformin therapy, insulin secretion
remains unchanged while fasting insulin levels and daylong plasma insulin
response may actually decrease.

Pharmacokinetics-

 Absorption –The absolute bioavailability of a metformin 500 mg tablet

administered in the fasting state is about 50%-60%. Single-dose clinical
studies using oral doses of metformin 500 to 1500 mg and 850 to 2550 mg
show that there is a lack of dose proportionality with an increase in
metformin dose, attributed to decreased absorption rather than changes in
elimination.
 Volume of distribution- The apparent volume of distribution (V/F) of
metformin after one oral dose of metformin 850 mg averaged at 654±385
L.
 Metabolism- Intravenous studies using a single dose of metformin in
normal objects show that metformin is excreted as unchanged drug in the
urine and does not undergo hepatic metabolism (no metabolites have been
identified in humans) or biliary excretion.
 Route of elimination- This drug is substantially excreted by the kidney.
Renal clearance of metformin is about 3.5 times higher than creatinine
clearance, which shows that renal tubular secretion is the major route of
metformin elimination. After oral administration, about 90% of absorbed
metformin is eliminated by the kidneys within the first 24 hours’ post-
ingestion.

Toxicity: Oral LD50 (rat): 1 g/kg; Intraperitoneal LD50 (rat): 500 mg/kg;
Subcutaneous LD50 (rat): 300 mg/kg; Oral LD50 (mouse): 1450 mg/kg;
Intraperitoneal LD50 (mouse): 420 mg/kg; Subcutaneous LD50 (mouse): 225
mg/kg
4 AIM AND OBJECTIVE

Aim: To Develop and Validate Stability Indicating RP-HPLC Method for

Simultaneous Estimation of Sitagliptin Phosphate Monohydrate and
Metformin Hydrochloride in Bulk and Marketed Formulation.

Objective –

To developed UV spectrophotometric method for Analysis sitagliptin

phosphate monohydrate and metformin Hydrochloride.
To developed HPLC method for simultaneous estimation of sitagliptin
phosphate monohydrate and metformin Hydrochloride bulk and tablet dosage
from.
To perform force degradation study.
To validated UV and HPLC method as per the ICH guidelines.
5. PLAN OF WORK

Plan of work divided into following steps:

1 Literature Review
2 Selection of drug molecule
3 Method development
4 Method validation as per ICH guidelines.
5 Force degradation study.
6 Preliminary analysis

5.1 Literature survey for metformin and sitagliptin regarding their

characteristics and analytical method.

5.2 UV Spectrophotometric method

 Selection of solvent
 Determination of scanning wavelength study of Beer- Lamberts law study
of additivity of absorbance at selected wavelength.

5.3 UV spectrophotometric method validation

 Performance the validation according to parameter of ICH guideline.

 Linearity
 Precision
 Accuracy
 Limit of detection
 Limit of quantitation
 Robustness
 Ruggedness
 Repeatability
5.4 Method development by RP-HPLC

 Selection of mobile phase.

 Selection of column.
 Selection of chromatographic conditions.
 Linearity range.
 System suitability parameters.
 Selectivity.
 Calibration curve and range.
 Accuracy and precision.
 Response factor.
 Stability.
 Recovery

5.5 Force degradation study

o Acid hydrolysis
o Alkaline hydrolysis
o Oxidation
o Thermal degradation
o Photo stability
 6. MATERIAL AND INSTRUMENTS
6.1 Procurement of drug sample
Table no.1. API and Supplier
Name of drug Quantity Supplier
Metformin 10gm Glenmark
hydrochloride Pharmaceutical Ltd.
Mumbai
Sitagliptin phosphate 5gm Glenmark
monohydrate Pharmaceutical Ltd.
Mumbai

6.2 Chemicals and reagents

All the chemicals used are of HPLC and AR grade. Chemicals used are as
follows.

Table no. 2. List of chemical and reagents

Sr.no Chemicals Grade

1. Acetonitrile HPLC
2. Water mili Q HPLC
3. Trimethylamine AR
4. Methanol HPLC
5. Potassium dihydrogen AR
phosphate
6. Orth phosphoric acid AR

6.3 Instruments
Sr. Instrument Make Model no.
no name
1.UV Spectrophotometer Shimadzu 1900
2.HPLC Agilent technology 1260-infinity II
3.Digital analytical balance Shimadzu AP225WD
4.Ultrasonicator Ultrasonic cleaner D- 120 /LH
5.Mili-Q Merck Millipore Direct-Q3
6 Infrared Bruker optics Alpha-T
spectrophotometer
7 PH meter Systronics MK VI
8 UV chamber Labin LV-28
Table no. 3. List of Instrument
7. EXPERIMENTAL WORK AND RESULT

7.1 Identification of drug

7.1.1Organoleptic properties of drug

Table no 4: Organoleptic properties of drug

Sr.no Organoleptic Metformin HCl Sitagliptin

property phosphate
monohydrate
1. Color white to off-white White to off
crystalline powder
compound
2. Odor Odorless Odorless

7.1.2 Melting point of drug

The melting point of a substance is the temperature at which it changes state
from solid to liquid. At the melting point the solid and liquid phase exist in
equilibrium.

Table no 5: melting point of drug

Sr.no Name of drug Observed M.P.(0C) Reference melting

point
1 Metformin HCl 2200C 219.0 to 223.0 °C

0
2 Sitagliptin phosphate 2040C 206.37 C
monohydrate

7.1.3 Solubility
Solubility of metformin Hydrochloride and sitagliptin phosphate monohydrate
was observed by dissolving them in different solvent and the observed results
are given in the table no.6
Table no 6: solubility study of metformin HCl and Sitagliptin phosphate
Sr.no Solvents Solubility
Metformin HCl Sitagliptin phosphate
monohydrate

1. Water Freely soluble Soluble

2. Methanol Soluble Slightly soluble
3. ACN Slightly soluble Soluble

4. NaOH Soluble soluble

7.1.4 FTIR spectrum of metformin hydrochloride
The IR study of pure was carried out by using Fourier infrared
spectrophotometer (BRUKER). Infrared absorption spectrum of metformin
and sitagliptin was recorded and interpreted over the wave Number 400-200
cm-1 using Fourier infrared spectrophotometer.
100.
0
99.
9
99.
8
Transmittance
99. [%] 99.
6 7
99.
5
3938.9

3887.7

3645.6

3474.0

3420.0

3369.3

3094.4

3029.4
2903.7

2783.8

2705.4

2519.2

2461.8

2413.0

2369.1
2096.8

2033.6

1844.8

1771.2

1619.4

1545.0

1441.3

1223.9

1157.5
1049.3

928.4

850.6

791.7
7

9
6

3500
3000 2500 2000 1500 1000
Wavenumber cm-1

C:\Program Files\OPUS_65\MEAS\METFORMIN METFORMIN HCL Solid 01/01/200

HCL.13 3

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Fig no:5 IR Spectrum of metformin Hydrochloride

Table no 7: Interpretation of FTIR spectrum of metformin Hydrochloride

Sr.no Functional group Standard range (cm- Observed range (cm-1)
1
)
1 C-H 2990-2850 2903.71
2 Primary and 3500-3150 3266.53-3474.09
secondary amine N-
H
3 C-N 1650-1600 1619.48
7.1.5 FTIR spectrum of sitagliptin phosphate monohydrate

100.
0
99.
9
Transmittance
99.
8
[%]
99.
7
99.
6
99.
5
3928.0

3851.5

3795.7

3739.2

3681.4

3567.3

3066.6

3007.6

2921.1

2723.7

2611.2

2515.1

2253.4

2173.9

2104.9

2027.7

1741.2

1678.5

1627.5
1417.0

1334.7

1140.3

972.6

868.3

724.6

679.8
1

0
3500
3000 2500 2000 1500 1000
Wavenumber cm-1

C:\Program Files\OPUS_65\MEAS\SITAGLIPTIN PHOSPHATE.0 SITAGLIPTIN PHOSPHATE Solid 01/01/200

3

Page 1/1

Fig no 6: IR Spectrum of sitagliptin phosphate monohydrate

Table no 8: Interpretation of FTIR spectrum of sitagliptin phosphate
monohydrate
Sr.no Functional group Observed Standard
range (cm-1) ranges
(cm-1)
1 C-F 1140.31-1334.77 1000-1400
2 C=O 1741.28 1700-1800
3 C-H 724.60-863.36 900-680
4 C=C 1534.94 1625-1440
5. N-H 3232.18-3468.25 3500-3150
6. C-H 2860.66-2921.17 2990-2850
7. C-N 1627.57 1650-1600
8. C-C 1263.68 1200

7.2 UV- Spectrophotometric Method

Development of simultaneous UV spectrophotometric method for metformin

hydrochloride and sitagliptin phosphate monohydrate.
7.2.1 Solvent selection: Different types Solvent were used for UV analysis,
among which water was selected due to separation and result there was no
derivation in the absorbance spectra.

7.2.2 Selection of wavelength: from standard stock solution, dilution were

prepared and scanned over the range of 200-400nm, the spectrum was
recorded and λmax observed for metformin HCl at 232 nm and sitagliptin
phosphate monohydrate at 266 nm.

7.2.3 Preparation of standard stock solution -

10 mg metformin HCl drug was accurately weighed and dissolved in 100ml of

distilled water in 100ml volumetric flask. (100µg/ml).

10mg sitagliptin phosphate monohydrate drug was accurately weighed and

dissolved in 10ml of distilled water in 10ml volumetric flask. (1000µg/ml).

Fig no7: UV spectrum of metformin Hydrochloride

Fig no 8: UV spectrum sitagliptin phosphate monohydrate

7.2.4 Validation of UV spectrophotometric method:

A) Linearity –

The proposed method was tested for linearity by plotting the absorbance
against concentration metformin HCl and sitagliptin phosphate monohydrate.
The developed method showed a linear response in concentration range of 2-
10µg/ml and 20-100µg/ml, correlation coefficient was calculated and found to
be 0.9992 and 0.9968. the result shows that and excellent correlation exist
between absorbance and drug concentration within the concentration range
indicated below.

Table no 9: Linearity data of Metformin Hydrochloride

Concentration (µg/ml) Absorbance

2 0.158
4 0.338
6 0.486
8 0.645
10 0.813
R2 0.999
y-intercept 0.0029
Slope 0.0808

Metformin Hydrochloride
1
y = 0.0808x + 0.0029
Absorbance

0.8
R² = 0.9992
0.6

0.4 abs

0.2 Linear (abs)

0
0 5 10 15
Concentration (µg/ml)

Fig no 9: Calibration curve of metformin Hydrochloride

 Table no 10: Linearity data of sitagliptin phosphate monohydrate
Concentration (µg/ml) Absorbance
20 0.079
40 0.167
60 0.234
80 0.325
100 0.388
R2 0.996
y-intercept 0.0058
Slope 0.0039

Sitagliptin phosphate monohydrate

0.5
0.4 y = 0.0039x + 0.0058
R² = 0.9969
Absorbance

0.3

0.2

0.1

0
0 20 40 60 80 100 120
Concentration (µg/ml)

Fig no 10: Calibration curve of sitagliptin phosphate monohydrate

B) Precision
Repeatability, interday, and intraday precision were assessed using three
concentrations and three replicates of each concentration. The % RSD was
found to be less than 2%, result shown in following table: 11-16.
Metformin HCl and sitagliptin phosphate monohydrate
1. Interday precision (metformin HCl)
 Table no 11: Precision data for metformin HCl (morning intraday)

Conc.(ppm) Absorbance Found % Mean

conc. Found %found
conc. conc.
2 0.162 1.9 98.45 99.84
0.163 1.9 99.07
0.168 2.0 102

4 0.333 4.0 102.5 100.7

0.328 4.0 100.5
0.324 3.9 99.3
6 0.482 5.9 98.8 100.6
0.491 6.0 100.6
0.500 6.1 102.5
Mean 100.4
SD 0.4703
%RSD 0.468

Table no 12: Precision data for sitagliptin phosphate monohydrate

(morning intraday)

Conc. Absorbance Found % Found Mean %

(ppm) conc. conc found
conc
20 0.084 20 100 98.4
0.081 19.2 96.4
0.083 19.7 98.9
40 0.159 39.2 98.2 99.43
0.163 40 100.7
0.161 39.7 99.4
60 0.239 59.7 99.6 99.73
0.238 59.5 99.2
0.241 60.3 100.4
Mean 99.1
SD 0.6975
%RSD 0.7033
Table no13: Precision data for metformin HCl (Afternoon intraday)

Conc. Absorbance Found conc %found Mean %

(ppm) conc found conc
2 0.156 1.8 94.7 94.5
0.156 1.8 94.7
0.155 1.8 94.1
4 0.318 3.8 97.4 96.9
0.316 3.8 96.8
0.315 3.86 96.5
6 0.478 5.8 97.9 98.1
0.480 5.9 98.6
0.477 5.8 97.7
Mean 96.5
SD 1.833
%RSD 1.899

Table no14: Precision data for sitagliptin phosphate monohydrate

(afternoon intraday)

Conc Absorbance Found conc %found Mean

no conc %found
(ppm) conc
20 0.081 19.2 96.4 96.8
0.083 19.7 98.9
0.080 19 95.1
40 0.156 38.5 96.2 97.1
0.159 39.2 98.2
0.157 38.7 96.9
60 0.237 59.2 98.8 99.2
0.239 59.7 99.6
0.238 59.5 99.2
Mean 97.7
SD 1.30
%RSD 1.33

2)Interday precision
Table no 15: Precision data for metformin HCl (morning interday)

Conc.no Absorbance Found conc %found Mean

(ppm) conc %found
conc
2 0.164 1.9 99.6 98.2
0.160 1.9 97.2
0.161 1.9 97.8
4 0.329 4.0 100 99.9
0.327 4.0 100
0.326 3.9 99.9
6 0.485 5.9 98.6 98.8
0.481 5.9 98.6
0.484 5.9 99.2

Mean 98.9
SD 0.86
%RSD 0.871

Table no 16: Precision data for sitagliptin phosphate monohydrate

(morning interday)

Conc Absorbance Found conc %found Mean %

no conc found conc
(ppm)
20 0.082 20.0 100 98.8
0.083 19.5 97.6
0.084 19.7 98.9
40 0.160 39.5 98.8 98.8
0.159 39.2 98.2
0.161 39.7 99.4
60 0.233 58.2 97.0 98.0
0.238 59.5 99.2
0.235 58.7 97.9
Mean 98.5
SD 0.461
%RSD 0.4687

C) Accuracy - The accuracy of the method studied at three different

concentration levels i.e., 80%,100%,120% showed affordable %recoveries in
the range of 94-98% and 96.25- 99.85%for metformin HCl and sitagliptin
phosphate monohydrate.
 Table no 17: Accuracy of metformin HCl (10ppm)

metformin HCl (10ppm)

% conc conc Conc Total Abs Found. Recovery SD %
of of conc conc % Found RS
sample API (ppm) ppm conc D
80% 1.0 0.8 18 1.388 17.14 95.23% 1.71 1.7
100% 1.0 1.0 20 1.588 19.6 98% 948 9%
120% 1.0 1.2 22 1.689 20.86 94.85%

Table no 18: Accuracy of sitagliptin phosphate monohydrate

Sitagliptin (20ppm)
% conc Tab Conc Total Abs Fond Recovery SD %RS
.conc of conc conc % found D
of API (ppm ppm conc
sample )
80% 2.0 1.6 36 0.146 35.94 99.85 1.864 1.89%
100% 2.0 2.0 40 0.156 38.5 96.28
120% 2.0 2.4 44 0.177 43.8 99

D) Robustness:

Robustness was studied by different deliberate variations in the UV

spectrophotometric conditions i.e. change in wavelength. From robustness study
% RSD was found to be within limit of 2% for the metformin HCl and
sitagliptin phosphate monohydrate. Hence it is robust and complies as per ICH
guidelines. Results are shown in table no. 19-22.
 Table no 19: Robustness data for metformin HCl 228nm

Metformin HCl 228nm

λmax Conc Absorbance Avg SD RSD %RS
waveleng (µg/ml)
R1 R2 R3 D
th
(nm)
228nm 2 0.187 0.188 0.18 0.187 0.001 0.005 0.53%
6 347
4 0.345 0.352 0.35 0.348 0.003 0.010 1.0%
1 5 0
6 0.493 0.495 0.51 0.499 0.009 0.018 1.86%
0 2 6

Table no 20: Robustness data for metformin HCl 236nm

Metformin HCl 236nm

λmax Conc Absorbance Avg SD RS %R
wavelengt (µg/ml R1 R2 R3 D SD
h )
(nm)
236nm 2 0.18 0.18 0.18 0.18 0.0005 0.00 0.31
5 6 5 5 7 31 %
4 0.34 0.35 0.35 0.34 0.0066 0.01 1.9
0 2 1 7 9 %
6 0.49 0.49 0.51 0.5 0.0087 0.01 1.74
4 6 0 74 %

Table no 21: Robustness data for sitagliptin phosphate monohydrate 263nm

sitagliptin phosphate monohydrate 263nm
λmax Conc Absorbance Avg SD RSD %RS
wavelengt (µg/ml R1 R2 R3 D
h )
(nm)
263nm 20 0.07 0.07 0.07 0.07 0.001 0.013 1.36%
3 2 4 3 6
40 0.16 0.16 0.16 0.16 0.0005 0.003 1.9%
1 2 1 1 7 5
60 0.25 0.26 0.26 0.26 0.0025 0.009 1.74%
9 4 2 1 1 6
 Table no 22: Robustness data for sitagliptin phosphate monohydrate 269 nm
sitagliptin phosphate monohydrate 269nm
λmax Conc Absorbance Avg SD RSD %RS
wavelengt (µg/ml R1 R2 R3 D
h )
(nm)
269nm 20 0.06 0.06 0.06 0.06 0.00115 0.018 1.81%
5 3 3 3 1
40 0.15 0.15 0.15 0.15 0.00057 0.003 0.37%
5 6 6 5 7 7
60 0.25 0.25 0.25 0.25 0.002 0.007 0.79%
1 5 3 3 9

E) Ruggedness
Conc Found conc. Avg SD RSD %RS
(µg/ml Absorbance D
)

R1 R2 R3 R1 R2 R3

2 0.15 0.15 0.15 1.8 1.8 1.9 0.15 0.00 0.019 1.9%
1 3 7 3 30
4 0.33 0.33 0.33 4 4.1 4.1 0.33 0.00 0.007 0.75%
3 8 5 5 25 5
6 0.48 0.48 0.48 5.9 6 5.9 0.48 0.00 0.006 0.61%
6 9 3 6 3 1
Table no 23: Ruggedness of metformin HCl

Co Absorbance Found conc. Avg SD RSD %RS

nc D
(µg
/ml R1 R2 R3 R1 R2 R3
)
20 0.07 0.080 0.077 18. 19.0 18.2 0.078 0.0015 0.019 1.9%
8 5 2
40 0.16 0.160 0.162 40 39.5 40 0.161 0.0015 0.009 0.94
3 4 %
60 0.23 0.237 0.236 57. 59.2 59.0 0.234 0.0037 0.016 1.61
0 8 8 1 %
Table no 24: Ruggedness of sitagliptin phosphate monohydrate
F) Limit of detection and limit of Quantitation
 LOD and LOQ values of metformin Hydrochloride and sitagliptin
phosphate
LOD – 3. 3×S.D
Slope
LOQ - 10×S.D
Slope

Table no 25: LOD and LOQ data for metformin HCl and Sitagliptin
phosphate

Drugs LOD(µg/ml) LOQ(µg/ml)

Metformin HCl 0.040(µg/ml) 0.123 (µg/ml)

Sitagliptin 0.846(µg/ml) 2.564(µg/ml)

phosphate
monohydrate
7.3 Method Development by RP-HPLC
7.3.1 Instrument and Apparatus

A RP-HPLC instrument 1260-infinity II with PDA detector, Open lab software

was used.

7.3.2 Selection of solvent

Water selected as solvent for determination of spectral characteristics of the

drug. The selection was based on the solubility of the drug in different
solvents.

7.3.3 Preparation of standard stock solution

Accurately weighed and transferred 10 mg of metformin Hydrochloride and 10

mg of sitagliptin phosphate drugs to the separate 10ml volumetric flasks and
added a diluent like water and sonicated to 10 minutes, after sonication make
up the volume up to 10 ml with same diluent.

7.3.4 Preparation of mobile phase A (pH 3.0 phosphate buffer):

 Dissolved 1.36g of potassium dihydrogen orthophosphate and 2ml of

trimethylamine in 800 ml of water, adjust the pH to 3.0 with
orthophosphoric acid and add sufficient water to produce 1000ml.
 Mobile phase was filtered through 0.22 µm membrane filter and degassed
by sonication for 30 min.

Preparation of mobile phase B: 100% Acetonitrile

Preparation of Diluent: Dilute with water up to mark

7.3.5 Selection of stationary phase

The chromatographic analysis was performed on X- Bridge C18 column

(4.6×250mm, particle size 5µm).

7.3.6 Selection of mobile phase

The composition and flow rate of mobile phase were changed to optimize the
separation condition using combined solution. Prepare mobile phase by taking
pH 3.0 phosphate buffer: Acetonitrile (65:35 %v/v). mobile phase was filtered
through 0.45µm membrane filter and degassed by sonication for 10 min. it was
established that the mobile phase pH 3.0 phosphate buffer: Acetonitrile (65:35
%v/v) shows good peak shape and resolution.

7.3.7 Optimization of mobile phase and chromatographic condition

For the RP-HPLC method development mobile phase was selected on the trial-and-
error basis. Method and some trial are reported as follow; the following
chromatographic condition were established by trial and error and were kept
constant throughout the method. The mobile phase were tried as follows: table no
28.

Table no 26: optimization of chromatographic condition

Sr.no Chromatographic condition Observatio

Mobile Column λmax Flow n
phase rate
1 Buffer : X- Bridge C18 210 1.0 Peak shape
ACN(55:45) column ml/mi was not
pH 3.0 (4.6×250mm, n good
particle
size 5µm)
2 Buffer : X- Bridge C18 210 1.0ml/ Peak shape
ACN(60 column min was good
:40) pH 3.0 (4.6×250mm, with
particle resolution
size 5µm) Problem
3 pH 3.0 X- Bridge C18 210 1.0ml/ Sharp peak
phosphate column min with good
buffer : (4.6×250mm,partical resolution,
Acetonitrile size 5µm) no. of
(65:35 theoretical
%v/v) plate
TRIAL-1:

Table No 27: Chromatographic condition for trial - 1

Parameters Conditions
Mobile phase Buffer : ACN(55:45) pH 3.0
Selection of column X- Bridge C18 column (4.6×250mm,
particle
size 5µm).
Injection volume 15µL
Column temperature Room temperature
Detection of wavelength 210nm
Flow rate 1.0ml/min
Conclusion Peak shape was not good

Fig no 11: chromatogram of trial 1

TRIAL-2:
Table no 28: chromatographic condition for trial 2

Parameters Conditions
Mobile phase Buffer : ACN(60 :40) pH 3.0
Selection of column X- Bridge C18 column
(4.6×250mm, particle
size 5µm).
Injection volume 15µL
Column temperature Room temperature
Detection of wavelength 210nm
Flow rate 1.0ml/min
Conclusion Peak shape was good with
resolution Problem

Fig no 12: chromatogram of trial 2

TRIAL-3:
Table no 29: Chromatographic condition for trial -3
Parameters Conditions
Mobile phase pH 3.0 phosphate buffer :
Acetonitrile (65:35 %v/v)
Selection of column X- Bridge C18 column
(4.6×250mm, particle
size 5µm).
Injection volume 15µL
Column temperature Room temperature
Detection of wavelength 210nm
Flow rate 1.0ml/min
Conclusion Sharp peak with good resolution, no.
of theoretical plate

<Chromatogram>

Fig no 13: chromatogram of trial 3

Observation: In the 3rd, pH 3.0 phosphate buffer: Acetonitrile (65:35 %v/v)

was selected mobile phase. It was observed that drug was eluted with good
resolution, theoretical plate, area which fulfilled acceptance criteria. Therefore,
chromatographic condition were optimized for the analysis.
Result: Method Accepted

A) Linearity

Metformin was found to bilinear in the concentration range of 5-25µg/ml and

sitagliptin is in the range of 10-50µg/ml. results obtained are shown in table
no. 32&33 and calibration plot obtained was shown in fig no.15&16 for
metformin and sitagliptin respectively.

Data of calibration curve of metformin and sitagliptin phosphate by

HPLC method

Sr.no Metformin HCl

Conc. Area
1. 5 857624
2. 10 1542003
3. 15 2411822
4. 20 3004280
5. 25 3919306
6. R2 0.9965
7. y-intercept 71315
8. Slope 151713
Table no 30: Linearity of metformin HCl

Metformin Hydrochloride
5000000

4000000 y = 151713x + 71315

R² = 0.9965
peak area

3000000

2000000 area

1000000 Linear (area)

0
0 10 20 30
Concentration µg/ml

Fig no 15: Calibration curve metformin HCl

 Sr.no Sitagliptin phosphate monohydrate
Conc Area
1 10 635603
2 20 1246610
3 30 1869809
4 40 2480078
5 50 3021238
6. R2 0.9994
7. y- intercept 49246
8 Slope 60047
Table no 31: Linearity of sitagliptin phosphate

Area
3500000
3000000 y = 60047x + 49246
R² = 0.9994
2500000
Peak area

2000000
1500000 area
1000000 Linear (area)
500000
0
0 20 40 60
concentration µg/ml

Fig no 16: Calibration curve sitagliptin phosphate monohydrate

B) Optical characteristics

Optical characteristics and statistical data of linearity for metformin HCl and
sitagliptin phosphate by HPLC method are summarized in table no. 32.
 Table no 32: Optical characteristics for metformin and sitagliptin

Sr.no Parameters Metformin Sitagliptin

HCl phosphate
monohydrate
1. λmax(nm) 210nm 210nm
2. Beers law limit 5-25 10-50
3. Regration equation[y] y = y =
151713x+71315 60047x+49246
4. Slope[m] 151713 60047
5. Intercept[c] 71315 49246
6. Correlation coefficient 0.9965 0.9994
[r2]
7. Limit of detection 0.0109µg/ml 0.499µg/ml
LOD(µg/ml)
8. Limit of quantitation 0.0330µg/ml 1.66µg/ml
(LOQ)(µg/ml)

C) Accuracy

Accuracy was studied by standard addition method and % recovery found was
within acceptable limit. Results of recovery study are show 98.8% to 99.5%
and 96.8%to99% of metformin HCl and sitagliptin phosphate monohydrate.
 50ppm of metformin HCl
% Conc Con Total Total Area Drug %reco
conc of c of Conc conc( recover very
mark stan (ml) ppm) ed
eted dard (µg/ml)
80% 5.0 4.0 9.0 90 13,636,264 89.4 98.8%
100% 5.0 5.0 10.0 100 15,172,428 99 99.5%
120% 5.0 6.0 11.0 110 16,691,749 109 99%
SD 0.360
RSD 0.0036
%RSD 0.36%

Table no 33: Accuracy of metformin HCl

5ppm of sitagliptin phosphate

%con Conc Conc Total Total Area Drug %
c. of of conc conc recover Recovery
mark stand (ml) (ppm) ed
eted ard (µg/ml)
80% 0.5 0.4 0.9 9 3,45359 4.9 98.6%
100% 0.5 0.5 1.0 10 6,47415 9.9 99%
120% 0.5 0.6 1.1 11 6,99810 10.8 96.8%
SD 1.154
RSD 0.0117
%RSD 1.17%

Table no 34: Accuracy of sitagliptin phosphate monohydrate

D) Precision

Intraday and interday precision assures the repeatability of test result. The %
RSD found was less than 2 for both metformin HCl and sitagliptin phosphate
monohydrate. Result of intraday and interday precision was shown in
respectively for metformin and sitagliptin was shown in table no.35 to table
no.38 respectively.

Precision study for metformin HCl

The %RSD found less than 2, hence results complies as per guidelines.

Sr. Conc. ( Area Found %found Mean SD RSD %R

no µg/ml) conc conc SD
1 5 829944 5.0 100% 82738 2703. 0.00 0.32
2 5 827642 4.9 99.7% 0.6 4 32 %
3 5 824556 4.9 99.2%
4 10 1559569 9.8 98% 15522 9445. 0.00 0.60

5 10 1556804 9.7 97% 792 27 60 %

6 10 1542003 9.6 96%

7 15 2311720 14.7 98% 23300 16599 0.00 0.71

8 15 2344125 14.9 99.8% 05 .39 71 %

9 15 2334172 14.9 99.4%

Table no 35: Data for intraday precision of metformin by HPLC method

Sr.n Conc Area Found %found Mean SD RS %R

o .(µg/ conc conc D SD
ml)
1 5 828845 4.9 99.8% 82858 1516. 0.00 0.18
2 5 829955 5.0 100% 5.3 26 18 %
3 5 826956 4.9 99.6
4 10 1545125 9.7 97% 15374 6794. 0.00 0.44
5 10 1535120 9.64 96.4% 68 41 44 %
6 10 1532159 9.6 96%
7 15 2314118 14.7 98.5% 23281 23390 0.01 1.0
8 15 2315125 14.7 98% 23 .7 0 %
9 15 2355126 15 100.3%
Table no 36: Data for interday precision of metformin by HPLC method
 Precision study of sitagliptin phosphate

The %RSD found less than 2, Hence results complies as per guidelines.

Table no 37: data for intraday precision of sitagliptin phosphate by HPLC

method

Sr.n Conc Area Found %fou Mean SD RSD %RSD

o (µg/m conc nd
l) conc
1 10 614976 9.4 94.2% 62045 5990 0.00 0.96%
2 10 626849 9.6 96.1% 0 .30 96
3 10 619525 9.4 94.9%
4 20 1233903 19.7 98.6% 12265 6712 0.00 0.54%
5 20 1224795 19.5 97.8% 01 .7 54
6 20 1220807 19.5 97.5%
7 30 1833164 29.7 99% 18419 9051 0.00 0.49%
8 30 1841421 29.8 99.4% 43 .81 49
9 30 1851245 30 100%
Table no 38:data for interday precision of sitagliptin phosphate by HPLC method

Sr Conc.( Area Found %fou Mean SD RSD %RS

.n µg/ml) conc nd D
o conc
1 10 634509 9.7 97.4 631667 5264.8 0.0083 0.83%
2 10 625603 9.5 95.9 7 4
3 10 634921 9.7 97.5
4 20 1232805 19.7 98 122692 5148.6 0.0041 0.41%
5 20 1223225 19.5 97.7 5
6 20 1224745 19.5 97.8
7 30 1829112 29.6 98.8 183634 8115.6 0.0044 0.44%
8 30 1845123 29.9 99.6 8
9 30 1834809 29.7 99.1
E) Robustness

Robustness was studied by different deliberate variations in the

chromatographic condition i.e. change in flow rate and wavelength. From
robustness study % RSD was found to be within limit of 2 % for the
metformin HCl and sitagliptin phosphate. Hence it is robust and complies as
per ICH guidelines. Results are shown in table no.39.

Sr. Parame con Metformin Sitagliptin

no ter diti HCl(10µg/ml) phosphate(20µg/ml)
on Area Mea SD %R Area Mea SD %RS
n SD n D
1 Change 0.8 1553 1234
in flow 159 910
2 rate 1 1531 1543 1122 0.72 1245 1243 723 0.58
(ml/min 120 366 2.5 % 796 105 5.8 %
3 ) 1.2 1545 1248
820 611
1 Change 206 1527 1220
in 350 134
2 wavelen 210 1599 1540 2209 1.43 1237 1225 103 0.84
gth 569 055 .5 % 162 259 41.0 %
3 (nm) 214 1527 1218 5
247 482
Table no 39: Robustness for metformin HCl and sitagliptin phosphate

F) Ruggedness

Ruggedness was studied by different analyst. From ruggedness study % RSD

was found to be within limit of 2% for the metformin HCl and sitagliptin.
Hence it is complying as per ICH guidelines. Results obtained are shown in
table no 40.
Sr. Analy Metformin HCl Sitagliptin phosphate
no st monohydrate
Area Mean SD %RS Area Mean SD %RS
D D
1 1559 12466
569 10
2 Analy 1548 15504 8406. 0.54 12339 12448 10228. 0.82
st –I 804 58 04 % 03 84 72 %
3 1543 12541
003 41
4 1525 12471
956 61
5 Analy 1527 15367 17638 1.14 12371 12433 5381.3 0.43
st- II 232 71 .6 % 62 11 %
6 1557 12456
125 10
Table no 40: Ruggedness for metformin HCl and sitagliptin phosphate

G) System suitability:

System suitability parameters were measured to verify the system, method and
column performance. Standard solution of metformin HCl and sitagliptin
phosphate monohydrate was injected into the system for five times and system
suitability parameters were checked.
 Table no 41: Data for system suitability study

Sr. Metformin HCl(10µg/ml) Sitagliptin phosphate

no. monohydrate (20µg/ml)
Retention Theoreti Asym Retentio Theoretic Asymme
time (min) cal metry n time al plates try
plates Factor (min) factor
1 2.260 2950 1.0 3.520 5833 1.15
2 2.26 2744 0.93 3.51 5469 1.14
3 2.267 2746 0.97 3.520 5632 1.13
4 2.260 2722 0.97 3.527 5600 1.11
5 2.260 2737 0.94 3.513 5540 1.14
Mean 2.2614 2735 0.96 3.52 5590 1.14
SD 0.0031 12.12 0.017 0.0054 46.7 0.01
%RS 0.138 0.44 1.80 0.155 0.83 0.87
D

H) Limit of detection and limit of quantitation

The results of LOD and LOQ are presented in table no.40

Table no 42: Results of LOD and LOQ values of metformin

Hydrochloride and Sitagliptin phosphate monohydrate.

Drugs LOD (µg/ml) LOQ (µg/ml)

Metformin 0.0109 µg/ml 0.0330µg/ml
HCl
Sitagliptin 0.499µg/ml 1.66µg/ml
phosphate
monohydrate
7.4 DEGRADATION STUDIES:

Stress testing of the drug substance can help to identify the likely degradation
products, the stability of the analytical procedure. Degradation studies were
performed on solutions containing metformin HCl (5µg/ml) and sitagliptin
phosphate (5µg/ml). Results of force degradation studies are summarized in
table no. 41&42.

7.4.1 photolytic degradation

Metformin Hydrochloride and sitagliptin phosphate drug was exposed to UV

light keeping the powder in a UV chamber for 24hrs. the solution was filtered
through 0.45µ syringe filter and injected under the chromatographic condition
and peak area was measured.

Fig no 17: Photolytic stressed chromatogram

7.4.2 Thermal degradation

Metformin Hydrochloride and sitagliptin phosphate powder was placed in an

oven at 500C for 24hrs. the solution was filtered through 0.45µ syringe filter
and injected under the chromatographic condition and peak area was
measured.
 Fig no 18: Thermal stressed chromatogram

7.4.3 Acid degradation

Metformin Hydrochloride and sitagliptin phosphate was subjected to forced

degradation by acidic hydrolysis using 0.1N HCl maintained at 60 0C for 1 hr.
and then the mixture was neutralized. The solution was filtered through
0.45µsyringe filter and injected under the chromatographic condition and peak
area was measured.

Fig no 19: Acid degradation

7.4.4 Alkaline degradation (Base)

Metformin Hydrochloride and sitagliptin phosphate was subjected to forced

degradation by acidic hydrolysis using 0.1N HCl maintained at 60 0C for 1 hr
and then the mixture was neutralized. The solution was filtered through
0.45µsyringe filter and injected under the chromatographic condition and peak
area was measured.

Fig no 20: Alkaline degradation(Base)

7.4.5 Oxidative degradation

Metformin Hydrochloride and sitagliptin phosphate was subjected to forced

degradation by hydrogen peroxide hydrolysis using 3% v/v H 2O2 at 600C for
30min. the solution was filtered through 0.45µ syringe filter and injected under
the chromatographic condition and peak area was measured.

Fig no 21: Oxidative degradation

 Table no 43: Forced Degradation data for metformin HCl

Sr.no Degradation Area of Area of Degraded Actual

standard degraded up to % %Degradation
sample
1 Acid 857624 803167 96.4784824 3.5215176
Degradation
2 Alkali 857624 756440 90.31856202 9.68143798
Degradation
3 H2O2 857624 635172 74.33206119 25.6679388
Degradation
4 Thermal 857624 807197 97.00974867 2.99025133
Degradation
5 Photolytic 857624 743267 88.58199363 11.4180064
Degradation

Table no 44: Forced Degradation data for sitagliptin phosphate monohydrate

Sr.no Degradation Area of Area of Degraded Actual %

standard degraded up to % degradation
sample
1 Acid 349763 291930 80.83134878 19.1686512
Degradation
2 Alkali 349763 149890 33.5217413 66.4782587
Degradation
3 H2O2 349763 295604 82.05505687 17.9449431
Degradation
4 Thermal 349763 327466 92.66741053 7.33258947
Degradation
5 Photolytic 349763 287153 79.97335421 20.0266458
Degradation
SUMMERY AND DISCUSSION:

Parameter Metformin Sitagliptin Acceptance

hydrochloride phosphate Criteria
Organoleptic characteristics
Color white to off-white White to off powder -
crystalline compound

Odor Odorless Odorless -

0
Melting 220 C 2040C
point
Absorbance 232nm 266nm -
maxima
UV Method Validation
Linearity
Range 2-10µg/ml 20-100µg/ml
Regression y = 0.0808x+0.0029 y = 0.0039x +
Equation 0.0058
Correlation 0.999 0.996 NMT0.99
Coefficient
Precision
Intraday Morning Intraday
precision
0.46 0.70 RSD NMT 2%
Afternoon Intraday
1.8 1.3
Interday Morning Interday
Precision
0.87 0.46
Accuracy
80% 1.79 1.89 RSD NMT 2%
100%
120%
Robustness Change in wavelength
236nm 269nm RSD NMT 2%
0.31 0.79
228nm 263nm
0.53 0.96
LOD 0.040 (µg/ml) 0.846 (µg/ml) -
LOQ 0.123 (µg/ml) 2.564(µg/ml) -
 RP-HPLC Method Validation
Linearity Metformin Hydrochloride Sitagliptin
Phosphate
Range 5-25µg/ml 10-50µg/ml -
Regression y = 151713x + 71315 y = 60047x +
Equation 49246

Correlation R² = 0.9965 R2=0.9994 NMT

Coefficient 0.99
System suitability Retention time 2.2min 3.5min
parameter Theoretical plate 2886 5629 >2000
Tailing factor 0.96761 1.131 Less
than 2.0
Resolution 7.07995 >2.0
Precision
Intraday precision Morning Intraday RSD
0.32 0.96 NMT
0.60 0.54 2%
0.71 0.49
Interday precision Morning Interday
0.18 0.83
0.44 0.41
1.0 0.44
Accuracy
80% 0.36 1.17 RSD
100% NMT
120% 2%
Ruggedness 0.54 0.82 RSD
1.14 0.43 NMT
2%
Robustness 206nm
0.72 0.58 RSD
214nm NMT
1.43 0.84 2%
LOD 0.0109µg/ml 0.499µg/ml -
LOQ 0.0330µg/ml 1.66µg/ml -
DISCUSSION:

The methods were developed and validated and satisfactory results were
obtained for most of the tests. The accepted method for RP-HPLC method has
data which showed good peak intensity, good retention time, theoretical plates,
asymmetry of the drugs. It can be easily and conveniently adopted for routine
quality control analysis. All methods are simple, rapid, accurate, precise and
reliable. Correlation coefficient for linearity of metformin hydrochloride and
sitagliptin phosphate by UV were 0.999 and 0.996. the linearity data for both
shoes excellent correlation between peak area and concentration of drug and it
is within the range. The relative standard deviation values for the methods
were less than 2%, which confirm the precision of the method.

The LOD and LOQ values of metformin hydrochloride and sitagliptin

phosphate by UV method were found to be 0.040(µg/ml),0.123(µg/ml) and
0.846(µg/ml),2.56 respectively. The LOD and LOQ values of metformin
hydrochloride and sitagliptin phosphate by RP-HPLC were found to be
0.0109µg/ml, 0.0330µg/ml and 0.499µg/ml, 1.66µg/ml respectively. The
method were validated as per ICH guidelines. The stress condition including
acidic, alkaline, oxidative, thermal, photolytic were applied.
CONCLUSION

In the present research work, a successful attempt was made for Development
and Validation of Stability Indicating RP-HPLC Methods for Estimation of
metformin Hydrochloride and sitagliptin in bulk and Pharmaceutical
Formulation. The method developed for analysis of metformin HCL and
sitagliptin phosphate monohydrate is simple, rapid, precise and robust. The
method was validated and satisfactory results were obtained for some of the
characteristics tested The system suitability parameters were selected for RP-
HPLC method and has data shown good peak intensity, good retention time
good Asymmetry of the drug that define the suitability method. It can be easily
and conveniently adopted for routine quality control analysis. All the analyzed
validation parameters showed acceptable data with satisfactory correlation co-
efficient and lower % RSD as per the ICH guidelines. The developed method
can be utilized by industry for quantitative simultaneous estimation of
metformin Hydrochloride and sitagliptin phosphate monohydrate as bulk and
in tablet dosage form.
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