K. Sujana et al /J. Pharm. Sci. & Res. Vol.

3(4), 2011,1122-1126

Difference Spectrophotometric Methods for Pioglitazone

Hydrochloride and Metformin Hydrochloride
K.Sujana, K.Abbulu, O.Bala Souri, B.Archana, M.Sindu, G.Swathi Rani
Department of Pharmaceutical Analysis, Acharya Nagarjuna University, Nagarjuna nagar-522510.
Abstract
Difference spectrophotometric method was developed for the estimation of Pioglitazone and Metformin in bulk drug
and in pharmaceutical formulations. Difference spectrum obtained, by keeping Pioglitazone and Metformin separately
in 0.1M NaOH in sample cell and 0.1M HCl as blank, showed characteristic peaks(λmax) at 228.1nm ( PIO) and
228.2nm (MET) and the characteristics peaks for pharmaceutical formulations were also found. This was depicted by
plotting the graphs between wavelength and absorbance. Difference of absorbance between these two maxima was
calculated to find out the amplitude, which was plotted against concentration. The optical characteristics for this method
are precise and accurate. The proposed method can be applied to pharmaceutical formulations and the common
excepients present in the formulation did not interfere with proposed method. The results indicate that the method is
simple, sensitive and can be used for routine estimation of Pioglitazone and Metformin in pharmaceutical dosage form.
Keywords: Difference spectrophotometry, Metformin, Pioglitazone

INTRODUCTION:
Pioglitazone and Metformin are the two new of 200-400nm throughout the experimental
anti-diabetic drugs and chemically Pioglitazone work.
is [(±)-5-[[4-[2-(5-ethyl-2-pyridinyl) ethoxy]
phenyl] methyl]-2,4-] thiazolidinedione Reagents and solutions:
monohydrochloride and Metformin is (N,N- Pioglitazone hydrochloride (PIO) and
dimethylimidodicarbonimidic diamide Metformin hydrochloride (MET) were gifted by
hydrochloride) . The main use for Pioglitazone and Matrix and Aurobindo Pharmaceuticals
and Metformin is in the treatment of diabetes respectively. Double distilled water was
mellitus type 2, especially in overweight prepared in the laboratory.
people. Difference spectrophotometry of
Pioglitazone and Metformin were not revealed Preparation of Solvents:
in literature. An attempt in the present study has 0.1M NaOH:
been made to develop simple, accurate and 2 gms of NaOH was accurately weighed and
economical method for difference spectroscopy dissolved in double distilled water and the
of Pioglitazone and Metformin and their volume was made up to 50ml with double
pharmaceutical formulations. The result of distilled water.
analysis using the developed 0.1M HCl:
spectrophotometric methods for difference 4.25ml of HCl was made up to 50ml with
spectrophotometry was found to be satisfactory double distilled water.
such that the developed methods can be used Phosphate Buffer PH-7:
for routine analysis of drugs and pharmaceutical 0.025gm of anhydrous disodium hydrogen
dosage form. phosphate and 0.0150gm of potassium
dihydrogen phosphate was accurately weighed
MATERIALS AND METHODS and dissolved in double distilled water and the
Apparatus volume was made upto 50ml with distilled
Spectral & absorbance measurements were water.
made on ELICO UV-Visible spectrophotometer Preparation of standard stock solution:
using 10mm Quartz matched cells with Accurately weighed 0.01gm (10mg) of PIO &
wavelength readability 0.1 nm increment was MET was dissolved in 10ml of ethanol in two
employed for all measurements. The absorption separate standard volumetric flasks to get
spectrum was recorded in the wavelength range 1mg/ml.

1122
 K. Sujana et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1122-1126

PROCEDURE: Fig.i.Absorbance maxima of PIO

For bulk samples:
From the standard solutions, 1ml of solution
was taken out and made up to 10ml with
ethanol and again from this 1ml was taken and
made up to 10ml with NaOH. From this 0.01,
0.02, 0.03, 0.04, 0.05 ml of aliquots of solution
were transferred into a series of 10ml
volumetric flasks and volume was made up to
10ml with NaOH. The amounts of PIO & MET
present in the sample were computed from their
calibration curves (using HCl as blank).Fig. vii
and viii.

For pharmaceutical preparations:

An accurately weighed tablet powder of Fig.ii.Absorbance maxima of MET
equivalent to 15mg of PIO & MET was
dissolved in 10ml of ethanol and filtered using
Whatmann filter paper. From the filtered
solutions, 1ml of each was taken and made up
to 10ml with NaOH and the absorbance was
checked using HCl as blank and the λmax of
pure drugs and formulations are plotted in
Fig.iii and iv.

Spectral Characteristics:
In order to ascertain the optimum wavelength of
the species formed, the spectra was scanned on
a UV-Visible spectrophotometer in the range of
200-400nm.λmax of acid, base and buffer was Fig.iii.Absorbance maxima of PIO pure drug
found separately. The base absorption spectra and Formulations
was identical with buffer, hence acid (HCl) was
used as a blank for base (NaOH) as PIO &
MET is basic in nature, base wavelength was
higher than acid as shown in Fig:i & ii.This is
because the absorption spectrum of PIO &
MET in acid medium shows hypsochromic shift
and hypochromic effect.
The absorption spectrum of PIO & MET was
measured by the absorbance of equimolar PIO
& MET solutions. The λmax of PIO & MET
was found to be 228.1nm and 228.2nm
respectively as shown is Fig.v and vi.

1123
 K. Sujana et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1122-1126

Fig.iv.Absorbance maxima of MET pure Fig.vii.Calibration of PIO

drug and Formulations

Fig.v.λmax of PIO Fig.viii.Calibration of MET

Sensitivity of the method:

Fig.vi.λmax of MET For finding the Beer’s limit of the method, the
absorbance for a set of solutions containing
varying amounts of PIO & MET was measured
at 228.1nm and 228.2nm against blank with
UV-visible spectrophotometer. The linearity
plot between absorbance and concentration of
PIO & MET showed that the system obeys
Beer’s law.
Precision of the method:
The precision of the method was established by
carrying out the analysis of the analytes (n=8)
using the proposed developed methods. The
low value of % Relative standard deviation
showed that the methods were precise. The
results are shown in Table. No.1 for PIO &
MET.

1124
 K. Sujana et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1122-1126

Accuracy of the method: maximum absorbance at 228.1nm for PIO and

To check the accuracy of the developed 228.2nm for MET. The correlation coefficients
methods and to study the interference of were found to be in between 0.999-1.0 which
formulation excipients, analytical recovery shows the good linear relationship for both
experiments were carried out by using standard components. The results of optical
addition method at 50, 100, 150% levels. From characteristics such as Beer’s law limits,
the total amount of drug found, the percentage correlation coefficient, slope, intercept and
recovery was calculated. The results revealed absorptivity coefficient values were
no interference of excipients. The results of summarized in Table-1.
recovery studies were summarized in Table -2 The tablet assay results obtained by proposed
for PIO & MET. methods were very close to labeled claim and
Optical characteristics such as Beer’s law low value of standard deviation, suggesting that
limits, molar extinction coefficient, correlation the developed methods has high precision. In
coefficient and regression equation, % relative order to check the accuracy of the developed
standard deviation (calculated from eight methods, known quantities of standard drugs of
measurements containing 3/4th of the amount of PIO & MET at three different levels were added
upper Beer’s law limits) were calculated and to its preanalyzed tablet sample and analyzed
results are shown in Table -1 for PIO & MET. by the developed methods. The results of
recovery studies are shown in Table-2. The
Table - 1: Optical characteristics of Pioglitazone mean percentage recoveries were found in the
and Metformin range of 98.0-101.3 shows no interference of
Parameters Pioglitazone Metformin the excipients in the tablet formulation.
Beer’s law limit (μg/ml) 0.1-0.5 0.1-0.5
max(nm) 228.1nm 228.2nm
Molar absorptivity
(L mole^-1 cm^-1)
77.566*10 57.049*10 CONCLUSION
Regression Y= (-0.25) Y=(-0.41) The data and information concerning drugs,
equation (y=b+aC) + 0.65C + 0.830C reagents and techniques given in results and
Slope (b) -0.25 -0.41 discussion reveal that the proposed methods are
Intercept (a) 0.657 0.830 simple, selective, sensitive (some are superior
Correlation
coefficient (r)
1.0 0.999 to the other methods) and accurate with
*Relative standard reasonable precision. In addition, selectivity to
0.52886 0.2790 each selected drug in its formulations was
deviation (%)
Y= b+aC, where ‘C’ is the concentration in mg/ml and achieved by selecting the appropriate
‘Y’ is the absorbance. combination of solvent systems, acids or bases
* calculated from eight replicate samples. in sample solution preparation and exploiting
specific functional groups exclusively present
Table - 2: Recovery Studies of Pioglitazone and
in the drug but not in the excepients, additives
Metformin:
Commercial **% Recovery of **% Recovery
or other active ingredient present in the
sample PIO of MET formulations, to the extent possible. The
Brand 1 98.0 99.8 proposed methods can be used as alternative
Brand 2 101.3 100.8 methods to reported ones and provide a wide
** Recovery amount was the average of 3 choice for the routine determination of the
determinations. above mentioned drugs depending upon the
availability of chemicals and situation arising
RESULTS AND DISCUSSION due to the presence of concomitants. The
The proposed method is based on difference proposed method is simple, sensitive, precise
spectrophotometric estimation of PIO & MET and accurate and can be used for routine
in UV region using ethanol as solvent. The estimation of Pioglitazone and Metformin in
absorbance spectral analysis shows the pharmaceutical dosage form.

1125
 K. Sujana et al /J. Pharm. Sci. & Res. Vol.3(4), 2011,1122-1126

ACKNOWLEDGEMENTS 5. Dunge, N. Sharda, B. Singh and S. Singh, J.

Authors are thankful for the Matrix and Pharm.Biomed. Anal., 2005, 37, 1109.
6. B. M. Rao, M. K. Srinivasu, G. Sridhar, P. R.
Aurobindo Pharmaceuticals for providing the Kumar,K. B. Chandrasekhar and A. Islam, J. Pharm.
gift samples of drugs Pioglitazone and Biomed. Anal., 2005, 39, 503.
Metformin. 7. L. Huber and S. George, eds., “Diode-array
detection in HPLC”, New York, Marcel Dekker,
REFERENCES 1993.
1. Indian Pharmacopoeia, Government of India 8. L. Huber, “Validation and qualification in
Ministry of Health and Family Welfare, Indian analytical laboratories”, Interpharm, 2002.
Pharmacopoeia Commission, Ghaziabad 3, 1674 9. R.T. Sane, S.N. Menon, Shafi Inamdar ,
(2007). Mandar Mote and Gunesh Gund, Simultaneous
2. United states Pharmacopoeia 30 NF 25 “The official Determination of Pioglitazone and Glimepiride by
compendia of standards, Asian edition, 2007, vol: 1. High-Performance iquid Chromatography ,
3. ICH Guidelines Q2BValidation of Analytical Chromatographia 2004, 451-453.
Procedures: Methodology (1996) 10. P. K. Sahoo, R. Sharma,* and S. C. Chaturvedi,
4. A.H.Beckett, J.B.Stenlake, PracticalPharmaceutical Simultaneous Estimation of Metformin
chemistry, IV-Edition, part two, CBS Publishers and Hydrochloride and Pioglitazone Hydrochloride by
distributors, 2004, p.no.326. RPHPLC Method from Combined Tablet Dosage
Form, Indian J Pharm Sci. 2008 May–Jun; 70(3):
383–386

1126