Development & Validation of Metformin & Emphagliflozine dosage forms by
Student: B.Jaffar Hussain
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
Established identity and purity.
Chapter 2: METHOD DEVELOPMENT AND
1 Introduction to Method Development
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
3 DRUG PROFILE
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
4 REVIEW OF LITERATURE
LAKSHMI et.al.. A
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.
Chapter: 5 NEED FOR THE STUDY
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.
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
PLAN OF WORK
To develop new RP HPLC method for the
simultaneous estimation of Metformin and empagliflozin pharmaceutical dosage form.
6.1 PLAN OF WORK
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
MATERIALS AND METHODS
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.
8.0 RESULTS AND DISCUSSION
8.1. Solubility Studies
studies are carried out at 25 0 C
soluble in methanol and in water, very slightly soluble in
Freely soluble in water,
soluble in acetonitrile,spraingly soluble in methanol.
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
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
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
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.
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.