NOVEL RP-HPLC METHOD FOR THE ... - Ijsidonline.info

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Thamma Narendra Kumar et al., IJSID, 2011, 1 (3), 395-421Where, TR = Metformin peak response from test preparation; SR = Metformin peak average response fromstandard preparation; W = Metformin Hydrochloride working standard weight in mg, for standard preparation; P =Metformin Hydrochloride working standard purity in %, on as such basis; L = Label claim of MetforminHydrochloride; A = Average weight of test sample and TW = Test weight taken in mg.RESULTS AND DISCUSSIONMethod development and optimizationIn pharmacopeial method for Metformin Hydrochloride assay by potentiometric titration method wasavailable. The main objective of this method development is to provide a suitable stability indicating assay methodfor quantification of Metformin Hydrochloride from different dosage forms. Method development trials wereperformed with different buffer salts, organic modifiers and columns. Finally the separation was achieved with Ionpairing buffer and acetonitrile with simple isocratic program (reported in materials and methods).Trial-1:The trial was performed using Waters Spherisorb SCX-10µ, 250 × 4.6 mm; 5.0 µm column. An isocraticelution of mobile phase is used, which is prepared by dissolving 17.0 g of Ammonium dihydrogen phosphate in 900ml of Acetonitrile and water mixture in the ratio 10:90, made up to 1000 ml with the same mixture and adjusted topH 3.0 with diluted Orthophosphoric acid. Then filtered the mobile phase through 0.45 µm PVDF filter anddegassed. The flow rate of the mobile phase was 2.0 ml/min. The column temperature was maintained at 25 o C andthe wavelength was monitored at 233 nm. The injection volume was 20 µl. The run time is fixed to 10 minutes.Diluent fixed as mixture of water and methanol in the ratio of 60:40.Observation:Blank interference was observed at the retention time of Metformin. Metformin peak and its impuritypeaks (Cyanoguanidine and Melamine) are not eluted at same retention time and concluded that can not beseparated by change in pH of the mobile phase.Trial-2:The second trial was performed using an Atlantis dC18, 250 × 4.6 mm; 5.0 µm column. The flow rate of themobile phase was 1.0 ml/min. The column temperature was maintained at 25 o C and the wavelength wasmonitored at 233 nm. The injection volume was 10 µl. The run time is fixed to 10 minutes. Diluent fixed as mixtureof water and methanol in the ratio of 60:40.An isocratic elution of mobile phase is used, which is a mixture of buffer (dissolved 2.2 g of 1-Heptanesulphonic acid sodium salt in 1000 ml of Milli-Q water and adjusted to pH 5.0 with diluted Orthophosphoric acid)and Acetonitrile.Trial-2(a):Taken mixture of buffer and acetonitrile in the ratio of 825 and 175 respectively. Then filtered the mobilephase through 0.45 µm PVDF filter and degassed.International Journal of Science Innovations and Discoveries Vol 1, Issue 3, November-December 2011398
Thamma Narendra Kumar et al., IJSID, 2011, 1 (3), 395-421Trial-2(b):Taken mixture of buffer and acetonitrile in the ratio of 800 and 200 respectively. Then filtered the mobilephase through 0.45 µm PVDF filter and degassed.Trial-2(c):Taken mixture of buffer and acetonitrile in the ratio of 750 and 250 respectively. Then filtered the mobilephase through 0.45 µm PVDF filter and degassed.Observation:From Trial-2(a) Metformin peak retention time was found more. With slight modification of composition inTrial-2(b) Metformin peak retention time was found lesser than previous trial. But in Trial-2(c) Metformin peakretention time was found less. Found no interference in blank and placebo chromatograms. All impurities werewell separated from active peak where as the peak shape, resolution and tailing factor were also within the limit.Different brand of <strong>HPLC</strong> columns were tried for getting the symmetrical peak of Metformin. In these trailsfound that Atlantis dC18, 250 × 4.6 mm; 5.0 µm column is the best suitable for the peak shape as well as forresponse of the Metformin. By this column obtained superior peak shape and separation because of the uniqueproperty of fully porous silica support with bonded phase of C12 with TMS end capping. This column is the bestsuite for the separation of basic compounds. Diluent was optimized based on the physical properties of the activesto get the better stability in the solution form with superior extraction of the actives from excipients in to thesolution form.Figure-2: Blank ChromatogramInternational Journal of Science Innovations and Discoveries Vol 1, Issue 3, November-December 2011399
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