METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS
ESTIMATION OF PIOGLITAZONE AND GLIMEPIRIDE – A UV
Kottu P. K*., Gadad A.P. and Dandagi P. M.
(Received 01 June 2012) (Accepted 12 october 2012)
The objective of the present work was to design a simple, accurate, economical and reproducible Uv spectrophotometric method for the simultaneous estimation of a two-component drug mixture of pioglitazone and glimepiride in the combined tablet dosage form. Methodology:
Simultaneous estimation method that involves maximum absorbance (λ max) of Pioglitazone and Glimepiride at 279.0 nm and 238.0 nm, respectively was developed. The proposed method was validated as per ICH guidelines for accuracy, precision, linearity, limit of quantification (LoQ) and limit of detection (LoD). The calibration curves were linear in the concentration range for pioglitazone (r value) and for glimepiride (r value) and were found to obey beer’s law in the linear concentration ranges. Statistical analysis and drug recovery data showed that simultaneous estimation method was simple, rapid, economical, sensitive, precise and reproducible. Hence, the proposed method was recommended for routine analysis of pioglitazone and glimepiride in combined tablet dosage form.
Pioglitazone, glimepiride, simultaneous
solid phase extraction method in human serum and
estimation, calibration curve and statistical
in dog serum as well as HPLC and LC mS in human
plasma have been reported for the estimation of pioglitazone. However the above mentioned methods
are sophisticated, expensive and time consuming
Chemically, pioglitazone (PIo) is (±) - 5- (4-
when compared to simple Uv spectrophotometric
[2- (5- ethyl- 2- pyridinyl) ethoxy] phenyl methyl]
- 2, 4- thiazolidinedione monohydrochloride
Glimepiride (GLIm) is a medium to long acting
(Fig. 1). It improves sensitivity to insulin in muscle and
sulfonylurea anti-diabetic drug. Glimepiride is a
adipose tissue and inhibits hepatic gluconeogenesis
potent third generation sulfonylurea derivative and
also improves glycemic control while reducing
is chemically, 3-ethyl-N
circulating insulin levels. It is used for the treatment
-pyrrol-2-yl) - 4-methyl-2-oxo-5H
of diabetes mellitus type 2 (previously known as
carboxamide (Fig. 2). It acts by stimulating insulin
non-insulin-dependent diabetes mellitus, NIDDm) in
secretions from the beta cells of pancreas and is
monotherapy and in combination with a sulfonylurea,
also known to increase peripheral insulin sensitivity,
metformin, or insulin. various analytical methods
thereby decreasing insulin resistance. Literature
like spectrophotometric method and HPLC and
survey reveals that few spectrophotometric, HPLC,
meCK method in tablet dosage form, HPLC and
HPLC-eSI – mS-mS and LCmS methods have been reported for the estimation of glimepiride 2.
* For correspondence
Department of Pharmaceutics,
Drugs are available in tablet dosage form as
KLE University’s College of Pharmacy
pioglitazone 30 mg and glimepiride 2 mg (Duetact)
Belgaum, Karnataka, India – 590010.
in the market. Literature survey revealed that
pioglitazone has been estimated with other drugs
using HPLC 3-8 and glimepiride has been determined
period of 15 min to remove any air bubbles. Similarly,
along with other drugs by Uv 9, and HPLC 6, 5-8, 10-11.
the standard stock solution of glimepiride (100 µg/ml)
Since no spectrophotometric method has been
was prepared. The stock solutions were individually
reported yet for simultaneous estimation of PIo
diluted with pH 2.0, HCl buffer containing 0.5% w/v
and GLIm in pH 2.0 HCl buffer an attempt has been
SLS to get final concentration of 20 µg/mL each
made to develop Uv-spectrophotometric method
and the diluted solutions were scanned in 200-400
for simultaneous estimation of pioglitazone and
nm range to determine the maximum absorbance
(λ max) of corresponding solutions 12. It was found that pioglitazone and glimepiride show maximum
MATERIALS & METHODS
absorbance (λ max) at 279.0 nm and 238.0 nm, respectively. All the solutions were filtered through
a 0.45µ membrane filter before they were scanned
A Shimadzu Uv/visible spectrophotometer,
model No. Uv-1700 was used for the study. The instrument had a spectral band width of 2 nm and
Preparation of standard solutions:
wavelength accuracy of ± 0.1 nm, with automatic
From the above prepared stock solution different
wavelength correction employing a pair of quartz cel s
aliquots of various concentrations (0.2, 1, 10, 25, 50,
of 1 cm patch length. A Denver electronic analytical
75 and 100µg/mL) were prepared using pH 2.0, HCl
balance (Tb214) was used for weighing the sample.
buffer containing 0.5 % w/v of SLS. The solutions
The pH of solutions was measured by a pH meter
were filtered through a 0.45µ membrane filter before
they were scanned by Uv Spectrophotometer for the linearity range. The linearity of the solutions was in
the concentration range of 1-12 µg/ml for Pioglitazone
Pioglitazone and glimipiride were kindly supplied
and Glimepiride, respectively. The Coefficient of
by Sun Pharma Limited, Jammu. Sodium lauryl
correlation for Pioglitazone and Glimepiride was found
sulphate (SLS) needles were obtained from S. D.
to be 0.9915 and 0.9904, respectively.
Fine Chem. Ltd, mumbai. Potassium dihydrogen phosphate was purchased from S. D. Fine Chem.
Simultaneous equation method:
Ltd, mumbai and sodium hydroxide was purchased
The simultaneous estimation of both pioglitazone
from Qualigens Fine Chemicals, mumbai. All the other
and glimepiride was done by simultaneous estimation
chemicals were of analytical grade. Double-distilled
method. Firstly, the absorptivity values of the both the
water was used throughout the study.
drugs were determined at λ of pioglitazone (279
nm, λ ) and glimepiride (238 nm, λ ). The absorptivity
both pioglitazone and glimipiride were tested for
value of the drugs is the ratio of absorbance at selected
purity by measuring its melting point and thin layer
wavelengths with the concentration of drugs in µg/ml.
chromatography for the presence of any impurities.
using the absorptivity values a set of two simultaneous equations were framed (eqs. 1 and 2).
The stock solution of the samples was further
Preparation of stock solutions
diluted with pH 2.0 HCl buffer containing 0.5 % w/v of
based on preliminary studies conducted for
SLS to get standard solution of concentration 10 µg/ml.
the solubilization of both the drugs, methanol was
The absorbance of the solution was measured at the
selected as suitable solvent for analysis. Firstly,
selected wavelengths and absorptivity was determined
standard stock solution (100 µg/ml) of pioglitazone
as a mean of three independent determinations.
was prepared by dissolving 10 mg of drug in 100 mL
Concentration of the drug in the samples was obtained
methanol. The solution was kept for sonication for a
and the calibration curve was plotted between
absorbance and concentration of the drug.
Inter-day and Intra-day Precision
Precision and accuracy was studied using solution
of concentration 10 µg/mL. Absorbance of the solution
was measured for three replicate samples. Intra-day
Where, A1 and A2 represent absorbance of
precision studies were run in triplicate on the same
mixture at λ1 and λ2, respectively, ax1 and ax2 are
day and inter-day on three consecutive days.
the absorptivities of PIo at λ1 and λ2 respectively and ay1, ay2 are the absorptivities of GLIm at λ1 and
Limit of Detection (LOD) and Limit of
λ2 respectively. Cx and Cy are the concentrations of
pioglitazone and glimepiride in µg/mL, respectively.
Validation of method
concentration of the analyte in the sample which can be analysed by the instrument. Limit of quantification
The method developed here was validated as per
(LoQ) is the minimum concentration of the analyte that
ICH guidelines 13-15 for its accuracy, linearity, precision, specificity, Robustness, limit of detection and limit of
can be reliably quantified. The Limit of detection (LoD)
and Limit of quantification (LoQ) were measured using following formula. Ten blank determinations
The accuracy of the developed method was
LoD = (3.3 * SD of analytical blank signals) / slope
determined by finding out the amount of recoveries
of pioglitazone and glimepiride. For the accuracy standard addition method was used where, as
LoQ = (10 * SD of analytical blank signals) / slope
known amounts of pioglitazone and glimepiride
of calibration curve. ------------- eq. 4
were added to the known concentration (10 µg/mL) of commercial tablets. The amount recovered was
Assay of tablet formulation by simultaneous
found by measuring the absorbance of the solution
and was expressed as mean recovery of samples
with upper and lower limits of percent relatives of
containing both pioglitazone and glimepiride
standard deviation. Recovery was done at three
(Duetact) were used for the study. Twenty tablets
different levels viz. 80%, 100% and 120%, within the
were taken and crushed. Powder equivalent to
10mg of PIo which includes 0.667 mg of GLIm and 9.33 mg of pure GLIm was added by standard addition
method in order to bring both drugs in 1:1 ratio and
The linearity of this method was evaluated by
the stock solution of this was prepared in methanol,
linear regression analysis and calculated by least
sonicated for 15 min, was then filtered through 0.45µm
square method and the drug shows linearity in the
membrane filter and then volume was made up to
concentration range of 1-12 µg/ml for both drugs.
100 ml with methanol. This stock solution contains
Standard dilutions were prepared using the required
100 µg/ml of each drug. Then the appropriate dilution
volume from the stock solution and then volume was
of 10 µg/ml was made using pH 2.0, HCl buffer
made up to 50 ml with pH 2.0, HCl buffer containing
containing 0.5% w/v SLS solution. All determinations
0.5% w/v SLS solution to yield the concentrations.
were carried out in triplicate. In simultaneous equation
Absorbance of the resulting solutions was measured
method, the absorbance of the prepared solutions
Table I: Optical Characteristics
Table II: Analysis of tablet formulation
Label claim (mg/tab)
Amount found (mg)
% drug found ± SD
Table III: Intra-day and Inter-day precision Pioglitazone and Glimepiride
Values expressed as mean of ± SD (n=3); SD: Standard deviation.
Table IV: Recovery
Amount added (µg/ml)
Amount recovered (µg/ml)
%Recovery ± SD
Values expressed as mean± SD (n=3); SD: Standard deviation
Table V: Limit of quantification (LOQ), Limit of detection (LOD)
Fig. 1: Chemical structure of Pioglitazone
Fig. 3: Overlain spectra of Pioglitazone and Glimepiride
Fig. 2: Chemical structure of Glimepiride
was observed at 279 nm and 238 nm and then the concentration of both the drugs was calculated using equation 1 and 2.
Fig. 4: Linearity graphs
drugs (Table I). Regression analysis was made for
The overlain spectrum (Fig. 3) of pioglitazone
the slope (m), intercept (c) and correlation coefficient
and glimepiride was found to be appropriate for the
(R) as shown in Table I. Higher values of correlation
estimation of both the drugs. based on the point of
coefficient (R) indicate good linearity of the calibration
maximum absorbance the λ of pioglitazone and
curve for both the drugs as shown in Fig. 4.
glimepiride was found to be 279 and 238 nm. The response for Pioglitazone was found to be linear
in the concentration range of 2 – 30 µg/mL at 279
determined by inter- and intra-day precision methods.
nm with correlation coefficient of 0.9904 (Table 1).
The results varied between 98.34 and 98.4 % in
Similarly, the derivative response for the glimepiride
case of inter-day precision for simultaneous equation
was found to be linear in the concentration range of
method while in case of intra-day precision it ranged
1 - 20 µg/mL at 238 nm with correlation coefficient
between 98.12%and98.57%. %RSD calculated was
found to be less than 2 indicating the accuracy and reproducibility of the method. Results are shown in
Amount of pioglitazone and glimepiride in
marketed formulation determined by the proposed method ranges between 98.80 and 99.20 %
The accuracy of the method was proved by
for simultaneous equation method as shown in
performing recovery studies on the commercial
formulation at 80, 100 and 120% level. Recovery range a from 99.50 to 100.83% in simultaneous
Linearity for detector response was observed
equation method (Table Iv). The results of recovery
in the concentration range of 1 – 20 µg/ml for both
study indicate that these drugs could be quantified
simultaneously and that there was no interference of
method forthe Determination of Pioglitazone in Rat Serum,
the excipients present in the formulation. The limits
International Journal of Pharmaceutical Sciences and
2011, 3(1), 38-41.
of detection and limit of quantification for pioglitazone
4. Srinivasulu D, Sastry b.S. and omprakash G.:
and glimepiride are shown in table v.
Development and validation of New Rp Hplc method for Determination of Pioglitazone HCl in Pharmaceutical
Dosage Forms, Int J Chem Res.
2010, 1(1), 18-20.
5. Lakshmi K.S, Rajesh T. and Sharma S.: Determination
The developed method involves formation and
of Pioglitazone and Glimepiride in Pharmaceutical
solving of simultaneous equation which is further
Formulations and Rat Plasma by Rp-Lc, International
based on absorptivity coefficients of two drugs at
Journal of Pharm Tech Research.
wavelength maxima of pioglitazone and glimepiride.
After the measurement of absorbance of sample
6. Havaldar Fh. and vairal Dl.: Simultaneous estimation of
Glimepiride, Rosiglitazone and Pioglitazone Hydrochloride
solution at both the wavelengths the amount of
in the Pharmaceutical Dosage Form, E-Journal of
drug in the sample can be found by substituting
2010, 7(4), 1326-1333.
the values in the two equations (eqs. 1 and 2). The
7. madhukar A, Naresh K, Kumar CH.N, Sandhya N. and
framed equation was validated as per ICH guidelines.
Prasanna P.: Rapid and Sensitive Rp-Hplc Analytical method
Statistical analysis and drug recovery data showed that
Development And validation of Pioglitazone Hydrochloride, Der Pharmacia Lettre
. 2011, 3(3), 128-132.
simultaneous estimation method was simple, rapid,
8. Karthik A, Subramanian G, mallikarjuna Rao C,
economical, sensitive, precise and accurate and can
bhat K, Ranjithkumar A, musmade P, Surulivelrajan
thereby be easily adopted for routine quality control
m, Karthikeyan K. and Udupa N.: Simultaneous
analysis. Results of this analysis confirmed that the
Determination of Pioglitazone and Glimepiride In bulk Drug and Pharmaceutical Dosage form by Rp-Hplc method,
proposed method was suitable for the simultaneous
Pak. J. Pharm. Sci
. 2008, 21(4), 421-425.
determination of these drugs in pharmaceutical
9. Khedekar P.b, Dhole S.m. and bhusari K.P.: Application
formulations with virtual y no interference of the
of vierodt’s and Absorption Correction Spectrophotometric
additives. Hence, the proposed method can be
methods for estimation of Rosiglitazone maleate and
successfully applied in simultaneous estimation of
Glimepiride in Tablets, Digest Journal of Nanomaterials
2010, 5(1), 77 – 84.
PIo and GLIm in marketed formulations.
10. Samala S, Tatipamula S.R. and veeresham C.:
Determination of Glimepiride in Rat Serum by Rp-Hplc
method, American Journal of Analytical Chemistry.
The authors are thankful to KLe College of
11. Khan I.U, Aslam F, Ashfaq m. and Asghar m.N.:
Pharmacy belgaum for providing al facilities to
Determination of glimepiride in pharmaceutical formulations
complete the work, Sun Pharma Limited, Jammu
using HPLC and first-derivative spectrophotometric
for providing pioglitazone and glimipiride, S. D.
methods, Journal of Analytical Chemistry.
Fine chem. Ltd, mumbai for providing sodium lauryl
12. Rubina R, bhagya Lakshmi J. and Ravi T.K.: Studies on
Formulation and In vitro evaluation of Glimepiride Floating
Tablets, C. J. Chem. Pharm. Res.
2011, 3(3), 159-164.
13. ICH Topic Q 2 (R1) validation of Analytical Procedures:
1. Shakya P. and Singh K.: Determination of Pioglitazone
Text and methodology, note for guidance on validation
Hydrochloride in bulk and Pharmaceutical Formulations
of analytical procedures: text and methodology (CPmP/
by Uv Spectrophotometric method, International Journal
of Pharmaceutical Sciences and Research.
14. International Conference on Harmonization; Draft
Guidance on specifications: Test Procedures and
2. mishra K, Soni H, Nayak G, Patel S.S. and Singhai A.K.:
Acceptance Criteria for New Drug Substances and
Development and validation of A Rp-Hplc method for
Products: Chemical Substances, Federal Register
estimation of Glimepiride in Tablet Dosage Form, IJPI’s
(Notices). 2000, 65(251), 83041–83063.
Journal of Analytical Chemistry
. 2011,1(3), 1-8.
15. FDA, Analytical Procedures and methods validation:
3. Ravikanth CH, Kumar A.A, Kiran v.U, Prashanth S,
Chemistry, manufacturing, and Controls, Federal Register
madhu b. and Reddy Y.N.: Sensitive and Rapid Hplc
(Notices). 2000, 65(169), 52,776– 52,777.
FDA NEWS RELEASE: PPI Warning For Immediate Release : May 25, 2010 Media Inquiries: Elaine Gansz Bobo, 301-796- FDA: Possible Fracture Risk with High Dose, Long-term Use of Proton Pump Inhibitors The U.S. Food and Drug Administration today warned consumers and health care professionals about a possible increased risk of fractures of the hip, wrist, and spine with high doses or long
Agenda der GPN-Studiensitzung in Halle, 17.11.2012, Beginn 9:00 Uhr Begrüßung, Protokollannahme und Bericht des Studienleiters Initial treatment of idiopathic nephrotic syndrome in children with mycophenolate mofetil vs. prednisone: A randomized, controlled, multicenter trial (INTENT Study) M. R. Benz, L. T. Weber (München), B. Tönshoff (Heidelberg) Für das Protokollk