International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 1, Issue 2, Oct-Dec. 2009 Research Article SIMULTANEOUS DETERMINATION OF METFORMIN AND PIOGLITAZONE BY REVERSED PHASE HPLC IN PHARMACEUTICAL DOSAGE FORMS
K.S. LAKSHMI1, T. RAJESH*1, SHRINIVAS SHARMA2
1Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Kattankulathur- 603203, Tamil Nadu, India.
2Department of Pharmacology, SRM College of Pharmacy, SRM University, Kattankulathur- 603203, Tamil Nadu, India.
Corresponding author e-mail: rajeshtirumala@hotmail.com
Ph. No-+91-442-745-3160, +91-995-201-4288
Received –8th June, 2009, Revised and Accepted – 4h August 2009
ABSTRACT:
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 (150x4.6mm, 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.
Key words: Metformin Hcl, Pioglitazone, Reverse phase HPLC, Pharmaceutical dosage forms INTRODUCTION
Metformin (I, N, N-dimethyldiguanide) and
determination of metformin and pioglitazone9-11
Pioglitazone, (±)-5-[p-[2-(5-ethyl-2-pyridyl)-
in pharmaceutical dosage forms. The present
ethoxy] benzyl]-2,4-thiazolidinedione1 are paper describes a simple, sensitive, validated
used in the treatment of type 2 diabetes.
and economic method for the determination
Metformin improves hepatic and peripheral
tissue sensitivity to insulin without the
MATERIALS AND METHODS
problem of serious lactic acidosis where as
Reagents
Pioglitazone hydrochloride has been shown to
Metformin and Pioglitazone were obtained
affect abnormal glucose and lipid metabolism
associated with insulin resistance by Mumbai, India. Acetonitrile and Methanol
enhancing insulin action on peripheral tissues.
Many patients suffering from type 2 diabetes
require treatment with more than one anti-
Instrumentation
hyperglycemic drug to achieve optimal The HPLC system consisted of a Shimadzu
The literature reveals that there are some of
the methods have been reported for detector. The data acquisition was performed
metformin UV1,2, HPLC3 stability studies4 and
potentiometry, spectrofluorimetry5. For Chromatographic conditions
pioglitazone HPLC method in pharmaceutical
dosage forms6 determination of its metabolites
RESULTS AND DISCUSSION
detector. The data acquisition was performed
by Spincotech 1.7 software. Analysis was
carried out at 255nm using a phenomenex C18
simultaneous determination of metformin and
reverse phase column of 150x 4.6 mm i.d., 5
pioglitazone in combined dosage form. The
µm dimensions at ambient temperature. The
chromatographic conditions were optimized
mobile phase consisted of Acetonitrile: by changing the mobile phase composition,
pH, and buffers used in the mobile phase.
ration of (42: 58, v/v) that was set at a flow
optimize the mobile phase. Finally a mixture
Preparation of stock and sample of Acetonitrile and Ammonium Acetate solutions
buffer (pH-3) in the ratio of 42:58 was used.
prepared with methanol to give the final
using the aforementioned mobile phase from
concentration of 1000 µg/ml. The working
20 µL of the assay preparation is illustrated in
Fig. 1. The retention times of MET and PIO
drug solution from the standard solutions
with mobile phase to get concentrations of
A mixed standard solution was prepared by
transferring 0.2 ml of each from the stock
(1000 µg/ml) into 10 ml volumetric flask and
made up the volume with mobile phase to get
For the analysis of pharmaceutical dosage
Fig. 1 : A typical chromatogram showing the peaks of metformin (5.17 min) and
tablet containing 500 mg of metformin HCl
pioglitazone (8.1 min) in pharmaceutical
and 30 mg of pioglitazone was transferred
dosage forms
into extraction flask, to this suitable amount
The linearity of the method was tested from
of methanol was added and the mixture was
0.5- 50 µg/ml for MET and 0.3- 30 µg/ml
subjected to vigorous shaking for 30 min
for PIO. Linearity solutions were injected in
for complete extraction of drugs, and then
triplicate and the calibration graphs were
centrifuged at 5000 rpm for 20 min (Remi
plotted as peak area of the analyte against
the concentration of the drug in µg/ml. In
the simultaneous determination, the calibration
Table 1 : Recovery of MET and PIO (n=3) Sample ID Concentration of drug (µg/ml) % Recovery Pure Drug Formulation MET PIO MET PIO MET PIO
the analytes in the mentioned concentrations
experiments. The recovery experiments were
and the correlation coefficients for the
performed by adding known amounts of the
regression line were 0.9968 and 0.9986 for
MET and PIO respectively. The accuracy of
determined at three levels, viz. 80%, 100%,
the method was studied by recovery and 120% of the selected concentrations. Table 2 : Precision data for MET and PIO Nominal concentrations (µg/ml) Mean±S.D, %RSD MET PIO MET PIO
25 1.5 24.67± 0.42, 1.75 1.46± 0.019, 1.30
50 3 48.76± 0.95, 1.95 2.89± 0.051, 1.76
100 6 98.35± 1.89, 1.92 5.86± 0.113, 1.93
Each mean value is the result of triplicate analysis for three times a day
Three samples were prepared for each recovery
specific for the quantitative determination of
level. The recovery values for MET and PIO
ranged from 98-102% and 97-103%, validation for different parameters, hence has
respectively (Table 1). The precision been applied for the estimation of drug in
(repeatability and intermediate precision) of the
pharmaceutical dosage forms. Tablets from
two different manufacturers (Piocon Forte
combined dosage form. Intra and Inter day
studies were performed by taking six replicates
Chemicals and Pharmaceuticals, and Diavista
of three concentrations. The results are shown
M MET 500 mg and PIO 15 mg, Dr. Reddy’s
in (Table 2). The limit of detection (LOD) and
Laboratories) were evaluated for the amount
limit of quantitation (LOQ) for MET, PIO was
of MET and PIO present in the formulations.
0.003 µg/ml, 0.0061 µg/ml and 0.01 µg/ml,
Each sample was analyzed in triplicate after
0.02 µg/ml, respectively. To determine the
extracting the drug as mentioned above in
robustness of the developed method experimental section. The amount of
experimental conditions were purposely altered
metformin and pioglitazone was found to be
and RSD of the peak areas of MET and PIO
within the range of 95%-105%. None of the
were found not greater than 2.0 illustrate the
tablet excipients were found to interfere with
the analyte peak and the results were shown
Application of the method to pharmaceutical dosage forms: The method is sensitive and Table 3 : Results of the determination of metformin and pioglitazone in Tablets (n=6) Labeled Amount (µg) Assay amount (mg) Taken Found±S.D %RSD %w/w CONCLUSION
The proposed method was found to be simple,
precise, accurate and rapid for simultaneous
determination of Metformin and Pioglitazone
forms. The mobile phase is simple to prepare
and economical. The sample recoveries in all
formulations were in good agreement with
Int. J. App Environ Sci 2008; 3: 65–73.
their respective label claims and they 5. Saad SM Hassan, Wagiha H Mahmoud,
suggested non-interference of formulation
excipients in the estimation. Hence, the
Metformin in Pharmaceutical Preparations
adopted for routine analysis of Metformin
and Pioglitazone in combined dosage forms
and UV–Visible Spectrophotometry, Anal
REFERENCES
Derivative Spectrophotometric Estimation
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Ultraviolet Spectrophotometry, Ind Drugs
Suresh B, Ion-Pair Liquid Chromatography
Extraction, J. Pharm. Biomed. Anal 1996;
Estimation of Pioglitazone hydrochloride
Determination of Pioglitazone and 11. Sahoo PK, Sharma R, Chaturvedi SC,
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