Simultaneous Estimation of Metformin Hydrochloride and Pioglitazone Hydrochloride by RPHPLC Method from Combined Tablet Dosage Form

A high performance reverse phase liquid chromatographic procedure is developed for simultaneous estimation of metformin hydrochloride and pioglitazone hydrochloride in combined tablet dosage form. The mobile phase used was a combination of acetonitrile:water:acetic acid (60:40:0.3) and the pH was adjusted to 5.5 by adding triethylamine. The detection of the combined dosage form was carried out at 230 nm and a flow rate employed was 1 ml/min. Linearity was obtained in the concentration range of 0.015 to 0.120 μg/ml of pioglitazone hydrochloride and 0.5 to 4.0 μg/ml of metformin hydrochloride with a correlation coefficient of 0.9992 and 0.9975. The results of the analysis were validated statistically and recovery studies confirmed the accuracy and precision of the proposed method.


Sahoo, et al.: RPHPLC Estimation of Metformin and Pioglitazone
A high performance reverse phase liquid chromatographic procedure is developed for simultaneous estimation of metformin hydrochloride and pioglitazone hydrochloride in combined tablet dosage form.The mobile phase used was *For correspondence E-mail: sunnitesh06@gmail.com a combination of acetonitrile:water:acetic acid (60:40:0.3)and the pH was adjusted to 5.5 by adding triethylamine.The detection of the combined dosage form was carried out at 230 nm and a fl ow rate employed was 1 ml/min.Linearity was obtained in the concentration range of 0.015 to 0.120 µg/ml of pioglitazone hydrochloride and 0.5 to 4.0 µg/ml of metformin hydrochloride with a correlation coeffi cient of 0.9992 and 0.9975.The results of the analysis were validated statistically and recovery studies confi rmed the accuracy and precision of the proposed method.
Key words: Pioglitazone hydrochloride, metformin hydrochloride, reverse-phase, simultaneous estimation Among the several mobile phases used for the simultaneous estimation of PIO and MET, acetonitrile:water:acetic acid (60:40:0.3)ratio was found to be most suitable and the pH was adjusted to 5.5 by adding triethylamine and was Þ ltered through 0.2 micron membrane Þ lter.
Standard stock solutions of PIO and MET were prepared by dissolving 5 mg of each in methanol and the volume were made up to 10 ml with mobile phase.From the above stock solutions dilutions were made in the concentration range of 0.015 to 0.120 µg/ml of PIO and 0.5 to 4.0 µg/ml of MET.All solutions were stored at room temperature.Each standard solution (20µl) was injected into the column after filtration using 0.2 micron membrane filter.All measurements were repeated five times and the calibration curves were constructed by plotting the peak area versus the corresponding drug concentration.The slope and correlation coefÞ cients were determined, which were found to be 0.9992 for PIO and 0.9975 for MET.
To determine the content of PIO and MET in tablet dosage form; twenty tablets were weighed; their average weight was determined and were finely powdered.Then 54.3 mg of triturate tablet dosage form was taken which is equivalent to 0.75 mg of PIO and was dissolved in 2 ml of methanol by stirring for 2 min.and the volume was made up to 10 ml using mobile phase.Then 1 ml from that solution was taken and diluted with mobile phase to make up to 10 ml.Again 2 ml from the later solution was taken and diluted with mobile phase to make up to 10 ml.The Þ nal solution was Þ ltered using 0.2-micron membrane filter and using an injection filter.Then with the help of 1000 µl micropipette 10, 20, 30, 40, 50, 60, 70, and 80 µl of the Þ ltered solution was taken in small test tubes and diluted up to 1000 µl of with the mobile phase, which contain 0.5:0.015,1.0:0.030,1.5:0.045,2.0:0.060,2.5:0.075,3.0:0.090,3.5:0.105,and 4.0:0.120µg/ml of both the drugs.20 µl of the above dilutions were injected one by one Metformin hydrochloride (MET) is an oral antidiabetic drug and is chemically N,Ndimethyl imidodicarbonimidic diamide.Few UV Spectrophotometric methods 1-2 , HPLC suitability tests are an integral part of chromatographic method.They are used to verify reproducibility of the chromatographic system.To ascertain its effectiveness, system suitability tests were carried out and its results are shown in Table 3.
to the HPLC with the help of Hamilton Syringe.The results are presented in Table 1.
The HPLC method was found to be simple, accurate, economic and rapid for routine simultaneous estimation of PIO and MET, in combined tablet dosage form.
The regression: 0.9992 and 0.9975, intercept:-222 and -3164 and slope: 671933 and 987374 were found to be for PIO and MET, respectively.Recovery was in the range of 99-101%; the value of standard deviation and percentage relative standard deviation were found to be less than 2%; shows the high precision of the developed method (Tables 1 and 2).
In proposed method, HPLC conditions were optimized to obtain, better separation of eluted compounds.Amongst the various mobile phases used, acetonitrile:water:acetic acid in 60:40:0.3ratio and the pH adjusted to 5.5 by the addition of triethylamine was found robust with 1 ml/min.ß ow rate.Mobile phase and flow rate selection was based on peak parameters such as height, tailing, theoretical plates, capacity factor, run time, resolutions etc.A typical chromatogram of PIO and MET is shown in (Þ g. 1).The optimum wavelength for detection was 230 nm at which detector response was best obtained.The average retention time for PIO and MET was found to be 5.35±0.05min and 2.150±0.05min, respectively.According to USP XXIV (621) 12 , system    Ondansetron combination with proton pump inhibitors has recently been introduced in market for the treatment of peptic ulcer, gastroesophagal reflux disease (GERD) and to prevent nausea.Ondansetron antagonizes 5HT-3 receptor both peripherally as well as centrally and block the initiation of the reflux, so is used as antiematic.Ondansetron is official in USP 1 .Omeprazole and rabeprazole are benzimidazole proton pump inhibitors, which suppress gastric acid

Fig. 1 :
Fig. 1: Typical Chromatogram of MET and PIO.Chromatogram showing retention time, 2.155 and 5.348 for MET and PIO in tablet dosage form, respectively.

TABLE 2 : RESULTS OF RECOVERY STUDIES
Qty.: Quantity.Results are mean of Þ ve replicates; percentage recovery is more than 99%.Hence the method is accurate and precise

TABLE 3 : RESULTS OF VALIDATION STUDIES
*Calculated at 5% peak height.SST: system suitability test, LOD: limit of detection, LOQ: limit of quantitation.