HPLC Method for Estimation of Metformin Hydrochloride in Formulated Microspheres and Tablet Dosage Form

A Shimadzu LC-10AT with SPD-10A detector was used for HPLC analysis. The reagents used for preparation of mobile phase were of HPLC grade. High performance liquid chromatographic method was developed using phenomenex C18 ODS (5 μ) 250 × 4.60 mm column, mobile phase selected for this method contains acetonitrile:phosphate buffer (65:35) pH adjusted to 5.75 with o-phosphoric acid which was filtered through 0.2 μ membrane filter. Flow rate employed was 1.0 ml/ min. Detection of eluent was carried out at 233.0 nm. Glipizide was used as the internal standard. Column was saturated with mobile phase for about an hour at above specified conditions. After the chromatographic conditions were set and the instrument was stabilized to obtain a steady base line a mixed standard dilution of pure drug containing 10 μg/ml of metformin hydrochloride and 5 μg/ml of glipizide (internal standard) were prepared in mobile phase, filtered through 0.2 μ membrane filter and loaded in the injector of instrument fitted with 20 μl fixed volume loop. The solution was injected three times and chromatogram recorded. The mean retention time for metformin hydrochloride and glipizide were found to be 2.30 and 3.95 min, respectively. The representative chromatogram of metformin hydrochloride and internal standard glipizide is reported in fig. 1.

Metformin hydrochloride chemically, N,Ndimethylimidodicarbonimidic diamide hydrochloride [1] is an antidiabetic agent [2] .In literature cited for analysis of metformin hydrochloride, few HPLC [3][4][5][6] methods have been reported but the retention time has been found to be very high and none have been reported from the microspheres.
A Shimadzu LC-10AT with SPD-10A detector was used for HPLC analysis.The reagents used for preparation of mobile phase were of HPLC grade.High performance liquid chromatographic method was developed using phenomenex C 18 ODS (5 µ) 250 × 4.60 mm column, mobile phase selected for this method contains acetonitrile:phosphate buffer (65:35) pH adjusted to 5.75 with o-phosphoric acid which was filtered through 0.2 µ membrane filter.Flow rate employed was 1.0 ml/ min.Detection of eluent was carried out at 233.0 nm.Glipizide was used as the internal standard.Column was saturated with mobile phase for about an hour at above specified conditions.After the chromatographic conditions were set and the instrument was stabilized to obtain a steady base line a mixed standard dilution of pure drug containing 10 µg/ml of metformin hydrochloride and 5 µg/ml of glipizide (internal standard) were prepared in mobile phase, filtered through 0.2 µ membrane filter and loaded in the injector of instrument fitted with 20 µl fixed volume loop.The solution was injected three times and chromatogram recorded.The mean retention time for metformin hydrochloride and glipizide were found to be 2.30 and 3.95 min, respectively.The representative chromatogram of metformin hydrochloride and internal standard glipizide is reported in fig. 1.
Standard stock solution of metformin hydrochloride and glipizide with concentration of 100 µg/ml was prepared separately in the mobile phase.For preparation of drug solutions for the calibration curve, in a series of 10 ml volumetric flask aliquots of standard stock solution of metformin hydrochloride 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 ml were transferred and to each flask 0.5 ml of glipizide standard stock solution was added and volume made up to the mark with mobile phase.Each solution was injected after filtration through 0.2 µ membrane filter and a chromatogram was recorded.The calibration curve was plotted between concentration of drug and ratio of peak area of metformin hydrochloride and glipizide (internal standard).Linearity was found to be in concentration range of 0 to 25 µg/ml of metformin hydrochloride with linear regression equation as y= 0.0204x+0.0012and the correlation coefficient value of 0.9990.
For analysis of formulation, formulated microspheres were accurately weighed and grounded to fine powder.Powder equivalent to 10 mg of metformin hydrochloride was accurately weighed and transferred to a 100 ml volumetric flask containing about 75 ml of mobile phase.The powder mixture was dissolved in the mobile phase with the aid of ultrasonication.The solution was filtered through Whatman filter paper No. 41 into another 100 ml volumetric flask washed the filter paper with mobile phase and added washings to the filtrate.Volume of the filtrate was made up to the mark with the mobile phase.From the above filtrate, 1 ml was taken in a 10 ml volumetric flask to which 0.5 ml of glipizide standard solution (100 µg/ml) was added and volume was made up to the mark with mobile phase, the solution was then filtered through 0.2 µ membrane filter.
After setting the chromatographic conditions and stabilizing the instrument, the formulated microsphere sample solution was injected and a chromatogram was recorded.The injection was repeated three times and the peak area of metformin hydrochloride and glipizide were recorded.The peak area ratio of drug to internal standard was calculated and the amount of drug present was estimated from the respective calibration curve.The analysis of formulated microspheres was compared by repeating the procedure on commercially available tablet formulation of metformin hydrochloride, the result of analysis is reported in Table 1.
To study the accuracy, reproducibility and precision of the above methods recovery studies were carried out by addition of standard drug solution to pre-analyzed sample of formulated microspheres at three different concentration levels (5, 10 and 15 µg).Results of recovery studies were found to be satisfactory and are reported in Table 1.
A HPLC method has been developed for estimation of metformin hydrochloride from pharmaceutical dosage form.The developed method for quantitative estimation of metformin hydrochloride from dosage form is reverse phase chromatographic method utilizing C 18 column and acetonitrile: phosphate buffer as mobile phase.Detection of the eluent was carried out using UV detector.The run time per sample is just 6 min.
The result of analysis of formulated microspheres using this developed method was found close to 100% for metformin hydrochloride, values of standard deviation was satisfactorily low indicating accuracy and reproducibility of the methods.Recovery studies were satisfactory and show that there is no interference of excipients.The developed methods were found to be simple, rapid, and accurate and can be used for routine analysis of metformin hydrochloride from tablet dosage form.In the traditional system of medicines e.g.Ayurveda, many of the remedies are in the form of powders.The dose of these powders is very large, many of them are difficult to swallow or give poor bioavailability owing to their poor wetting and many have pungent or unacceptable taste.It is proposed in Ayurveda that ideally dosage form should be pleasantly tasted and in a compact form [1] .One of the possible approaches to overcome these drawbacks of Ayurvedic powders is to present them in the form of a unit dosage form i.e. tablet.Triphala powder is widely used as anti diabetic and for eyesight improvement [2] in Indian population and has poor compressibility index (40%).Hence Triphala powder was selected as a model for investigation of binding and granulating properties of hydrogel isolated from whole seeds of Ocimum basilicum.Compressibility can be imparted by preparing granules following wet granulation technology and by using suitable granulating and binding agent [3] .In an attempt to avoid complexities associated with development of synthetic and semi synthetic excipients, an unexplored area of herbal products was tried.The whole seeds of Ocimum basilicum were authenticated by Agharkar Research Institute, Pune.The hydrogel [4] was isolated from whole seeds and used as a binding and granulating agent to prepare granules of Triphala powder.The seeds were soaked in deminerised water in a proportion 1:20 for 2 hrs.Then the whole mass

Fig. 1 :
Fig. 1: Chromatogram of metformin hydrochloride and the internal standard glipizide

Bhosale,
et al.: Hydrogel Isolated from Seeds of Ocimum basilicum as Binder Ayurvedic powders are widely used as therapeutic agents but most of them have unpleasant taste and large doses.One of the possible approach to overcome these drawbacks is to represent them in unit dosage form i.e. tablet dosage form.The purpose of this study is to elucidate and quantify the compressibility and compactibility of herbal granules prepared by using hydrogel isolated from whole seeds of Ocimum basilicum as a novel binder.The compressibility is the ability of the powder to deform under pressure and the compactibility is the ability of a powder to form coherent compacts.To test the functionality of novel excipients, Sonnergaard proved a simple linear model to confirm compactability, which is an uncomplicated tool for quantification.The tablets were compressed at increasing compression pressures and were evaluated for various mechanical properties.The linear relationship between specific crushing strength and compression pressure revealed the compactibility of the herbal granules and the linear relationship between porosity and logarithm of compression pressure revealed the compressible nature of the herbal granules according to the model developed by Sonnergaard.Thus the hydrogel isolated from whole seeds of Ocimum basillicum had potential as a granulating and binding agent.Key words: Ocimum basilicum, compressibility and compactibility, novel binder, hydrogel

Investigation of Hydrogel Isolated from Seeds of Ocimum basilicum as Binder
A. V. BHOSALE*, S. HARDIKAR, A. A. PATHAK AND R. V. SABLE Poona District Education Association's Seth Govind Raghunath Sable College of Pharmacy, Saswad, Pune-412 301, India