Method drug substance with inert ingredient (excipient)

Development & Validation of Metformin & Emphagliflozine dosage forms by


Student: B.Jaffar Hussain 


Guige G.Somasekhar





analysis simply means analysis of pharmaceuticals. Webster’ dictionary defines
a pharmaceutical is a medical drug. A more appropriate term for a
pharmaceutical is active pharmaceutical ingredient (API) or active ingredient
to distinguish it from a formulated product or drug product is prepared by
formulating a drug substance with inert ingredient (excipient) to prepare a
drug product that is suitable for administration to patients. Research and
development (R) play a very comprehensive role in new drug development
and follow up activities to ensure that a new drug product meets the
established standards is stable and continue to approved by regulatory
authorities ,assuring that all batches of drug product are made to the specific
standards utilization of approved ingredients and production method becomes the
responsibility of pharmaceutical analysts in the quality control (QC) or
quality assurance department . The methods are generally developed in an
analytical R department and transferred to QC or other departments as
needed. At times they are transferred to other divisions.

By now it should
be quite apparent that pharmaceutical analysts play a major role in assuring
the identity, safety, efficacy, and quality of drug product, safety and
efficacy studies required that drug substance and drug product meet two
critical requirements.

Established identity and purity.

Established bio


Analytical chemistry

A branch of chemistry that deals
with the identification of compounds and mixtures (qualitative analysis) or the
determination of the proportions of the constituents (quantitative
analysis).The techniques commonly used are titration, precipitation,
spectroscopy, chromatography, etc

Analytical chemistry serves the
needs of many fields:

ü  In industry, analytical chemistry provides the
means of testing raw materials and for assuring the quality of finished
products whose chemical composition is critical. Many household products,
fuels, paints, pharmaceuticals, etc. are analyzed by the procedures developed
by analytical chemists before being sold to the consumer.

ü  The nutritional value of food is determined by
chemical analysis for major components such as protein and carbohydrates and
trace components such as vitamins and minerals. Indeed even the calories in a
food are often calculated from its chemical analysis.

ü  In medicine, analytical chemistry is the basis
for clinical laboratory tests, which help physicians to diagnose disease and
chart the progress in recovery.

ü  Environmental quality is often evaluated by
testing for suspected contaminants using the techniques of analytical

chemists also make important contributions to fields as diverse as forensic
chemistry, archaeology, and space science1.

1.2  Chromatography:

is a family of analytical chemistry
techniques for the separation of mixtures.
It involves passing the sample, a mixture that contains the analyte,
in the “mobile phase”, often in a stream of solvent,
through the “stationary phase.” The stationary phase retards the
passage of the components of the sample. When components pass through the
system at different rates they become separated in time, like runners in a
marathon. Ideally, each component has a characteristic time of passage through
the system. This is called its “retention time.”

physical separation method in which the components of a mixture are separated
by differences in their distribution between two phases, one of which is
stationary (stationary phase) while the other (mobile phase) moves through it
in a definite direction. The substances must interact with the stationary phase
to be retained and separated by it.

chromatograph takes a chemical
mixture carried by liquid
or gas
and separates it into its component parts as a result of differential
distributions of the solutes
as they flow around or over a stationary liquid or solid phase. Various
techniques for the separation of complex mixtures rely on the differential
affinities of substances for a gas or liquid mobile medium and for a stationary
medium through which they pass; such as paper,
or magnesium silicate gel. Analytical
chromatography is used to determine the identity and concentration of
molecules in a mixture. Preparative
chromatography is used to purify larger quantities of a molecular


1.3.1 Adsorption Chromatography

Adsorption chromatography is probably
one of the oldest types of chromatography around. It utilizes a mobile liquid
or gaseous phase that is adsorbed onto the surface of a stationary solid phase.
The equilibration between the mobile and stationary phase accounts for the
separation of different solutes.

1.3.2 Partition Chromatography

form of chromatography is based on a thin film formed on the surface of the
solid support by a liquid stationary phase. Solute equilibrates between the
mobile phase and the stationary liquid.

1.3.3 Ion Exchange Chromatography

this type of chromatography, the use of a resin (the stationary solid phase) is
used to covalently attach anions or cations on it. Solute ions of the opposite
charge in the mobile liquid phase are attracted to the resin by electrostatic

   1.3.4 Molecular Exclusion Chromatography

known as gel permeation or gel filtration, this type of chromatography lacks an
attractive interaction between the stationary phase and solute. The liquid or
gaseous phase passes through a porous gel, which separates the molecules
according to its size. The pores are normally small and exclude the larger
solute molecules, but allow smaller molecules to enter the gel, causing them to
flow through a larger volume. This causes the larger molecules to pass through
the column at a faster rate than the smaller ones.

1.3.5 Affinity Chromatography

is the most selective type of chromatography employed. It utilizes the specific
interaction between one kind of solute molecule and a second molecule that is
immobilized on a stationary phase. For example, the immobilized molecule may be
an antibody to some specific protein. When this molecule passes solute
containing a mixture of proteins, only the specific protein is reacted to this
antibody, binding it to the stationary ph This protein is later extracted by
changing the ionic strength or pH.


is able to separate macromolecules and ionic species, labile natural products,
polymeric materials and a wide variety of other high-molecular weight poly
functional groups.

  1.4.1 Basic principle of HPLC

Ø  High
performance liquid chromatography (HPLC) is a separation technique utilizing
differences in distribution of compounds to two phases; called stationary phase
and mobile phase.

Ø  The
stationary phase designates a thin layer created on the surface of fine
particles and the mobile phase designates the liquid flowing over the
particles. Under a certain dynamic conditions, each component in a sample has
difference distribution equilibrium depending on solubility in the phases and
or molecular size.

Ø  As
a result the components move at different speeds over the stationary phase and
are thereby separated from each other.

Ø  The
column is a stainless steel (or resin) tube, which is packed with spherical
solid particles.

Ø  Mobile
phase is constantly fed into the column inlet at a constant rate by a liquid
pump. A sample is injected from a sample injector located near the column

Ø  The
injected sample enters the column with the mobile phase and the components in
the sample migrate through it passing between the stationary and mobile phases.

Ø  Compound
move in the column only when it is in the mobile phase. Compounds that tend to
be distributed in the mobile phase therefore migrate faster through the column
while compounds that tend to be distributed in the stationary phase migrate

Ø  In
this way each component is separated on the column and sequentially elutes from
the outlet. A detector connected to the outlet of the column detects each
compound eluting from the column.


i. Based on
modes of chromatography

Normal phase chromatography

Reverse phase chromatography

ii. Based on
principles of separation

Adsorption chromatography

Ion exchange chromatography

Ion pair chromatography

Size exclusion chromatography

Affinity chromatography

Chairal phase chromatography

iii. Based on
elution technique

Isocratic separation

Gradient separation

iv. Based on the
scale of operation

Analytical HPLC

Preparative HPLC3-7

Normal Phase High Performance Liquid Chromatography (NP-HPLC)

Normal-phase liquid-liquid
chromatography uses a polar stationary phase and less polar mobile phase. To
select an optimum mobile phase, it is best to start with a pure hydrocarbon
mobile phase such as heptanes. If the sample is strongly retained, the polarity
of the mobile phase should be increased, perhaps by adding small amounts of
methanol or dioxane.

In the normal phase mode, separations of
oil-soluble vitamins, essential oils, nitro phenols, or more polar homologous
series have been performed using alcohol/heptanes as the mobile phase. Column
used in normal phase chromatography for chiral separation: Chiracel OJ and
Chiracel OD.

 1.5.2 Reverse Phase High Performance Liquid
Chromatography (RP-HPLC)

     Reverse phase chromatography uses hydrophobic
bonded packing, usually with an octadecyl or octyl functional group and a polar
mobile phase, often a partially or fully aqueous mobile phase.

     Polar substances prefer the mobile phase
and elute first. As the hydrophobic character of the solutes increases,
retention increases.

     Generally, the lower the polarity of the
mobile phase, the higher is its eluent strength. The elution order of the
classes of compounds in table is reversed (thus the name reverse-phase




1 Introduction to Method Development

            The number
of drugs introduced into the market is increasing every year. These drugs may
be either new entities or partial structural modification of the existing one.
Often a time lag exists from the date of introduction of a drug into the market
to the date of its inclusion in pharmacopoeias. This happens because of the
possible uncertainties in the continuous and wider usage of these drugs, reports
of new toxicities (resulting in their withdrawal from the market), development
of patient resistance and introduction of better drugs by competitors. Under
these conditions, standards and analytical procedures for these drugs may not
be available in the pharmacopoeias. It becomes necessary, therefore to develop
newer analytical methods for such drugs. 11,12

                Analytical methods should be used
within good manufacturing practice (GMP) and good laboratory practice (GLP)
environments, and must be developed using the protocols set out in the
International Conference on Harmonization (ICH) guidelines (Q2A and Q2B). 13,14







is a sodium glucose co-transporter-2 (SGLT-2) inhibitor
indicated as an adjunct to diet and exercise to improve glycemic control in
adult patients with type 2 diabetes. SGLT2 co-transporters are responsible for
reabsorption of glucose from the glomerular filtrate in the kidney. The
glucuretic effect resulting from SGLT2 inhibition reduces renal absorption and
lowers the renal threshold for glucose, therefore resulting in increased
glucose excretion. Additionally, it contributes to reduced hyperglycaemia and also
assists weight loss and blood pressure reduction.


Used in Diabetes

Tract and Metabolism

Glucose Lowering Drugs, Excl. Insulins

Weight:  450.91

Chemical Formula:






simple, sensitive and rapid reverse phase high performance liquid
chromatographic method was developed for the estimation of Metformin Hcl (MET)
and Pioglitazone (PIO) in pure and in pharmaceutical dosage forms. A Gemini C18
column (150×4.6mm, 5µ) was used with a mobile phase containing a mixture of
Acetonitrile and Ammonium Acetate buffer (pH-3) in the ratio of 42: 58. The
flow rate was 0.3ml/min and effluents were monitored at 255nm and eluted at
5.17min (MET) and 8.1min (PIO). Calibration curve was plotted with a range from
0.5-50 µg/ml for MET and 0.3-30 µg/ml for PIO. The assay was validated for the
parameters like accuracy, precision, robustness and system suitability
parameters. The proposed method can be useful in the routine analysis for the
determination on metformin and pioglitazone in pharmaceutical dosage forms.

et al.. In the present study, two analytical methods were developed for
the estimation of Dapagliflozin in API. Method A: RPHPLC method, Method B: UV
spectroscopic method. In method A, the drug showed linearity in the range of
25-150µg/ml with a correlation coefficient (r2 ) of 0.999, where as in method
B, the linearity range was found to be 1-5µg/ml with a correlation coefficient
of (r2 ) 0.999. Both the methods were validated for different validation
parameters such as linearity, accuracy, precision, detection limit, quantitation
limit, robustness and ruggedness and the results were found to be within the
acceptance limits as per the guidelines of International Conference on
Harmonization (ICH).

K. Y et al.. A simple, RP-HPLC method was established for
determining linagliptin and metformin in pharmaceutical formulations.
Linagliptin , metformin and their degradation products were separated using C8
column with Acetonitrile: Water: Methanol (25:50:25 (v/v/v) to pH 4.1 with 0.1%
orthophosphoric acid as the mobile phase. Detection was performed at 243 nm
using a diode array detector. The method was validated using ICH guidelines and
was linear in the range 5-30µg/ and 10-100 µg /ml for linagliptin and metformin
respectivily. Good separation of both the analytes and their degradation
products was achieved using this method. The developed method can be applied
successfully for the determination of linagliptin and metformin.







5.0  Analytical Method Development for Pharmaceutical Formulations

Quality investigation plays a very important role in
quality specification establishment of chemical drugs. The number of drugs introduced into the market every year .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. Hence, standards and
analytical procedures for these drugs may not be available in the
pharmacopoeias. It becomes necessary, therefore to develop newer analytical
methods for such drugs.

Basic criteria
for new method development of drug analysis:

The drug or drug combination may
not be official in any pharmacopoeias.

A proper analytical procedure for
the drug may not be available in the literature due to patent regulations.

Analytical methods may not be
available for the drug in the form of a formulation due to the interference
caused by the formulation excipients.

Analytical methods for a drug in
combination with other drugs may not be available.

The existing analytical procedures
may require expensive reagents and solvents. It may also involve cumbersome
extraction and separation procedures and these may not be reliable.

Analytical method development provides the support to
track the quality of the product from batch to batch.


Chapter: 6 AIM AND



6.0   AIM

 To develop new RP HPLC method for the
simultaneous estimation of Metformin and empagliflozin  pharmaceutical dosage form.




Solubility determination of Metformin and
empagliflozin various solvents and buffers.


Determine the absorption maxima of both the
drugs in UV–Visible region in different solvents/buffers and selecting the
solvents for HPLC method development.


Optimize the mobile phase and flow rates for
proper resolution and retention times.


Validate the developed method as per ICH



Chapter: 7

7.4. Mobile Phase:

A mixture of 50 volumes
of methanol and 50 volumes of  phosphate
buffer were prepared. The mobile phase was sonicated for 10min to remove gases
and filtered through 0.45µ membrane filter for degassing of mobile phase.


7.4.1. Preparation of Mixed Phosphate

1.625 gm of
potassium di hydrogen
phosphate (KH2PO4)and 0.3 gm of Dipotassium hydrogen phosphate was weighed and
dissolved in 100ml of water and volume was made up to 550ml with water. Adjust
the pH to 4.0 using ortho phosphoric acid. The buffer was filtered through
0.45µ filters to remove all fine particles and gases.





8.1. Solubility Studies

studies are carried out at 25 0 C


soluble in methanol and in water, very slightly soluble in
phosphate buffer.


Freely soluble in water,
soluble in acetonitrile,spraingly soluble in methanol.

8.2. Determination
Of  Working Wavelength (?max)

In simultaneous estimation of  two drugs isobestic wavelength is used.
Isobestic point is the wavelength where the molar absorptivity is the same for
two substances that are interconvertible. So this wavelength is used in
simultaneous estimation to estimate both drugs accurately.

8.2.1. Preparation of standard
stock solution of METFORMIN

mg of METFORMINwas
weighed and transferred in to 100ml volumetric flask and dissolved in methanol
and then make up to the mark with methanol and prepare 10 µg /ml of solution by
diluting 1ml to 10ml with methanol.

8.2.2. Preparation of standard
stock solution of EMPAGLIFLOZIN

 10mg of EMPAGLIFLOZINwas weighed in to 100ml
volumetric flask and dissolved in Methanol and then dilute up to the mark with
methanol and prepare 10 µg /ml of solution by diluting 1ml to 10ml with

8.2.3. Results

           The wavelength of maximum absorption
(?max) of the drug, 10 ?g/ml solution of the drugs in methanol were
scanned using UV-Visible spectrophotometer within the wavelength region of
200–400 nm against methanol as blank. The resulting spectra are shown in the
fig. no. 8.1, 8.2 and 8.3 and the absorption curve shows characteristic
absorption maxima at 240 nm for METFORMIN, 229 nm for EMPAGLIFLOZIN and
255nm for the combination.


Chapter: 9.0 VALIDATION


9.1 System

Standard solutions were
prepared as per the test method and injected into the chromatographic system.
The system suitability parameters like theoretical plates, resolution and
asymmetric factor were evaluated.

The % RSD for the retention times of METFORMIN and EMPAGLIFLOZIN Peaks
from 6 replicate   injections of each
Standard solution should be not more than 2.0 %

2.         The % RSD for the peak area responses of
METFORMIN and EMPAGLIFLOZIN peaks from 6 replicate injections of each standard
solution should be not more than 2.0%. 

3.   The number of theoretical plates (N) for the METFORMIN
and EMPAGLIFLOZIN peaks is not less than

  The Tailing factor (T) for the METFORMIN
and EMPAGLIFLOZIN peak is not more than 2.0.  


The % RSD for the
retention times and peak area of METFORMIN and EMPAGLIFLOZIN were found to be
less than 2%. The plate count and
tailing factor results were found to be satisfactory and are found to be within
the limit. 



Chapter: 10 Discussion


                 A simple and selective LC
method is described for the determination of Metformin and
empagliflozin in tablet dosage forms. Chromatographic separation was achieved on
a c18 column using mobile phase consisting of a mixture of 50
volumes of methanol and 50 volumes of 
phosphate buffer with detection of 255 nm. Linearity was
observed in the range 60-140 µg /ml for Metformin (r2
=0.997) and 3-7µg /ml for empagliflozin (r2
=0.995) for the amount of drugs estimated by the proposed methods was in good
agreement with the label claim.

                The proposed methods were
validated. The accuracy of the methods was assessed by recovery studies at
three different levels. Recovery experiments indicated the absence of
interference from commonly encountered pharmaceutical additives. The method was
found to be precise as indicated by the repeatability analysis, showing %RSD
less than 2. All statistical data proves validity of the methods and can be
used for routine analysis of pharmaceutical dosage form.




11 Conclusion


From the
above experimental results and parameters it was concluded that, this newly
developed method for the simultaneous estimation Metformin and
empagliflozindrugs was found to be simple, precise, accurate and
high resolution and shorter retention time makes this method more acceptable
and cost effective and it can be effectively applied for routine analysis in
research institutions, quality control department in meant in industries,
approved testing laboratories studies in near future.




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