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Pak. J. Pharm. Sci., Vol.21, No.4, October 2008, pp.421-425 421
SIMULTANEOUS DETERMINATION OF PIOGLITAZONE AND GLIMEPIRIDE
IN BULK DRUG AND PHARMACEUTICAL DOSAGE FORM
BY RP-HPLC METHOD
KARTHIK A*, SUBRAMANIAN G, MALLIKARJUNA RAO C,
KRISHNAMURTHY BHAT, RANJITHKUMAR A,
MUSMADE P, SURULIVELRAJAN M, KARTHIKEYAN K AND UDUPA N
Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal, India
ABSTRACT
A simple, fast, and precise reverse phase, isocratic HPLC method was developed for the separation and
quantification of pioglitazone and glimepiride in bulk drug and pharmaceutical dosage form. The quantification
was carried out using Inertsil ODS (250 × 4.6 mm, 5µ) column and mobile phase comprised of acetonitrile and
ammonium acetate (pH 4.5; 20mM) in proportion of 60:40 (v/v). The flow rate was 1.0 ml/min and the effluent
was monitored at 230 nm. The retention time of pioglitazone and glimepiride were 7.0±0.1 and 10.2±0.1 min
respectively. The method was validated in terms of linearity, precision, accuracy, and specificity, limit of
detection and limit of quantitation. Linearity of pioglitazone and glimepiride were in the range of 2.0 to
200.0µg/ml and 0.5-50µg/ml respectively. The percentage recoveries of both the drugs were 99.85% and
102.06% for pioglitazone and glimepiride respectively from the tablet formulation. The proposed method is
suitable for simultaneous determination of pioglitazone and glimepiride in pharmaceutical dosage form and
bulk drug.
Keywords: Pioglitazone, Glimepride, HPLC, method validation.
INTRODUCTION
Pioglitazone is a thiazolidine Dione derivative. It is one of
the PPAR-alpha agonist, insulin sensitizer used to reduce
the insulin resistance. Pioglitazone (fig. 1) is chemically
[(±)-5-[[4-[2-[5-ethyl -2- pyridinyl) ethoxy] phenyl]-
methyl]-2,4-]thiazolidinedionemonohydro-chloride. Gli-
mepiride is a sulfonylurea urea derivative chemically-[[p-
[2-(3-ethyl-4-methyl-2-oxo-3-pyyroline-1-oxamide)ethyl]
phenyl] sulfonyl]-3-(trans-4-methylcyclohexyl) urea,
widely used in patients with type 2 diabetes (non-insulin-
dependent diabetes). The drugs are prescribed
individually as well as multi component dosage forms
available in the market. A number of methods have been
published for the estimation of the above said analytes.
Pioglitazone in human plasma (Venkatesh et al., 2007;
Xue et al., 2006 and Sripalakit et al., 2006) and HPLC
method for antidiabetic drugs (Yao et al., 2006; Jedlicka
et al., 2004 and Kolte et al., 2004) were reported. Method
for estimation of glimepiride in human plasma
(Chakradhar et al., 2007; Saleem et al., 2004) was
published. HPLC method for estimation of glimepiride
and related substances (Deep et al., 2005; Kovarikova et
al., 2004; Khan et al., 2005) were also reported in the
literature. Even though various methods were reported in
the literature for estimation of glimepiride and
pioglitazone individually or in combination with other
drugs no method had been reported for simultaneous
estimation of these two drugs using HPLC in bulk drug
and pharmaceutical dosage forms. The present study was
aimed at the simultaneous estimation of pioglitazone and
glimepiride by reverse phase HPLC method. The method
was validated according to the ICH (Q2A 1995)
guidelines.
EXPERIMENTAL
Materials, reagents and chemicals
Pioglitazone and Glimepiride were obtained as gift
samples from Dr. Reddys Laboratories, Hyderabad.
Ammonium acetate and glacial acetic acid were A.R
grade from SD fine chemicals Mumbai. acetonitrile
HPLC grade from Merck chemicals, Mumbai.
Chromatographic condition: Water alliance 2695-
separation module with waters 2487 dual UV detector
was used. Millennium software version 4.0 s used for
Data acquisition .Inertsil ODS (25 cm × 4.6 mm, 5µ)
column was used as a stationary phase. Mobile phase
comprised of acetonitrile and 20mM ammonium acetate
buffer (60; 40 v/v) with pH adjusted to 4.5±0.2 was used.
Injection volume was 10µl and run time was 12min and
flow rate 1.0ml/min. The column was maintained at
ambient temperature and the eluent was detected at 230
nm.
Solutions
Standard solutions
Standard stock solution (1000 µg/ml) of pioglitazone,
glimepiride were prepared separately in methanol. The
*Corresponding author: Fax: +91-820-2571998, e-mail: millinkarthik@yahoo.co.in
Simultaneous determination of pioglitazone and glimepiride in bulk
Pak. J. Pharm. Sci., Vol.21, No.4, October 2008, pp.421-425
422
working standard solutions were prepared and further
diluted in mobile phase to contain a mixture of
pioglitazone and glimepiride in over the linearity range
from 2-200 µg/ml and 0.5-50 µg/ml respectively.
Assay in formulations
Twenty tablets, Pioryl, (Panacea Biotech), each
containing 15mg of pioglitazone and 2mg of glimepiride
were weighed and finely powdered. A quantity of powder
equivalent to 15mg of pioglitazone and 2mg of
glimepiride was weighed and transferred to a Standard
flask. The drug was diluted using methanol to get a
concentration of 10µg/ml of pioglitazone, 1µg/ml of
glimepiride. The contents were mixed thoroughly and
filtered through a 0.45 µ filter. 10µ of the sample was
injected in to HPLC system.
RESULTS AND DISCUSSION
The proposed HPLC method required fewer reagents and
materials, and it is simple and less time consuming. This
method could be used in quality control test in
pharmaceutical industries. The chromatograms of
pioglitazone and glimepiride were shown in (fig. 4).
There was clear resolution between pioglitazone and
glimepiride with retention time of 7.0 and 10.2 minutes
respectively.
Validation of the method
Linearity
The response for the detector was determined to be linear
over the range of 2 to 200µg/ml (2, 5, 10, 25, 50, 100,
200) for pioglitazone and 0.5-50µg/ml (0.5, 1, 2, 5, 10,
25, 50) for glimepiride. Each of the concentration was
injected in duplicate to get reproducible response. The
calibration curve was plotted as concentration of the
respective drug versus the response at each level. The
proposed method was evaluated by its correlation
coefficient and intercept value calculated in the statistical
study. They were represented by the linear regression
equation (figs. 2, 3).
Y Pioglitazone = 27494X+32335, ‘r’ value= 0.9995
Y Glimepiride = 37719X-3261, ‘r’ value= 0.9987
Slopes and intercepts were obtained by using regression
equation (y=mx+c) and least square treatment of the
results used to confirm linearity of the method developed.
Precision and accuracy
The accuracy of the method was determined by recovery
experiments. The recovery studies were carried out 6
times and the percentage recovery and % relative standard
deviation was calculated. From the data obtained,
recoveries of standard drugs were found to be accurate
(table 1).
The %CV of interday and intraday precision obtained was
less than 1% for both the drugs. The intraday and interday
precision of pioglitazone was 0.47 and 0.86 and
glimepiride was 0.76 and 0.94 respectively. From the data
obtained, the developed HPLC method was found to be
precise and accurate.
Specificity of the method
The PDA chromatograms of the pioglitazone and
glimepiride in standard and sample were recorded. In the
chromatograms of the formulations, some additional
peaks were observed which may be due to excipients
present in the formulations. These peaks however did not
interfere with the standard peaks, which demonstrate that
the assay method is specific. Furthermore, the purity of
the peaks was studied by peak purity studies. The results
revealed that the peak is free from interferences, which
shows that the HPLC method is specific.
Quantification limit
The limit of detection (LOD) and limit of quantification
(LOQ) of the developed method determined by injecting
progressively low concentrations of the standard solutions
using the developed methods. The LOD is the lowest
concentration of the analyte that can be detected with
signal to noise ratio (1:3) and LOQ is the lowest
concentration that can be quantified with acceptable
precision and accuracy with signal to noise ratio (1:10).
The LOD of pioglitazone and glimepiride found to be
0.2µg/ml and 0.1µg/ml respectively. The LOQ of
pioglitazone and glimepiride found to be 2µg/ml and
0.5µg/ml respectively
Table 1: Recovery studies
Name of the Drug Spiking level
(%) Amount added
(µg/ml)
Amount
recovered
(µg/ml) n=3
Percentage
recovery
Average
percentage
recovery
80 8 7.89 98.63
100 10 10.21 102.1
Pioglitazone 120 12 11.86 98.83
99.85
80 0.8 0.82 102.5
100 1 1.02 102
Glimepiride 120 1.2 1.22 101.67
102.06
Karthik A et al.
Pak. J. Pharm. Sci., Vol.21, No.4, October 2008, pp.421-425 423
Robustness
The robustness of the method was studied by deliberate
changes in the method like alteration in pH of the mobile
phase, percentage organic content, changes in the
wavelength. It was observed that there was no marked
changes in the chromatograms demonstrate that the HPLC
methods have developed are robust.
Solution Stability
In this study, the mobile phase, the standard solutions, and
the sample solution were subjected to long term (3 days)
stability studies. The stability of these solutions was
studied by performing the experiment and looking for
changes in separation, retention, and asymmetry of the
peaks which were then compared with the pattern of the
chromatogram of freshly prepared solutions.
System suitability
The resolution, capacity factor, theoretical plates/meter,
Rt values and peak symmetry were calculated for the
standard solutions. The values obtained demonstrated the
suitability of the system for the analysis of the above drug
combinations System suitability parameters might be fall
within ± 3% standard deviation range during routine
performance of the method. The summary of the method
validation results were showed in the (table 2).
Table 2: Summary of analytical method validation
Validation parameters Pioglitazone Glimepiride
Recovery (%) 99.85 102.06
Intraday precision (%CV) 0.47 0.76
Interday precision (%CV) 0.86 0.94
Linearity (r2) 0.9995 0.9987
Robustness (%CV) 0.66 0.82
LOD(µg/ml) 0.2 0.1
LOQ ( µg/ml) 2 0.5
Specificity Passed Passed
(a)
(b)
Fig. 1: Chemical structures of (a) Pioglitazone (b) Glimepiride
0
1000000
2000000
3000000
4000000
5000000
6000000
0 50 100 150 200 250
Conc(µg/mL)
Peakarea
Fig. 2: Calibration curve of pioglitazone
Simultaneous determination of pioglitazone and glimepiride in bulk
Pak. J. Pharm. Sci., Vol.21, No.4, October 2008, pp.421-425
424
CONCLUSION
This method is simple, specific and easy to perform and
requires short time to analyze the samples. Low limit of
quantification and limit of detection makes this method
suitable for use in quality control. This method enables
simultaneous determination of Pioglitazone and
Glimepiride because of good separation and resolution of
the chromatographic peaks. The method was found to be
accurate, precise, linear, robust and rugged.
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