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Thamma Narendra Kumar et al., IJSID, 2011, 1 (3), 395-421Response (mAU)Robustness:9000000800000070000006000000500000040000003000000200000010000000Linearity Plot for Metformin Hydrochloride0.000 20.000 40.000 60.000 80.000 100.000 120.000 140.000 160.000 180.000Concentration (µg/ml)Figure-49: Linearity graphsTo determine the robustness of the developed method, experimental conditions were deliberately altered.The flow rate of the mobile phase was 1.0 ml/min, to study the effect of flow rate; flow was changed by 0.1 unitsfrom 0.9 to1.l ml/min. The effect of the column temperature was studied at 20 and 30 o C instead of 25 o C. The effectof the mobile phase organic composition was studied using 90% and 110% organic phase composition instead of100% organic phase composition. The effect of the buffer pH in mobile phase was studied using mobile phase withpH 4.90 buffer and pH 5.10 buffer instead of mobile phase with pH 5.00 buffer. The effect of filters was conductedusing two different filters namely, 0.45µm PVDF filter and 0.45µm Nylon filter.In all the deliberate varied chromatographic conditions (flow rate, column temperature, mobile phaseorganic composition and buffer pH in mobile phase), the tailing factor of Metformin peak was less than 2.0 and thetheoretical plate count was more than 2000. 0.45 µm PVDF filter and 0.45 µm Nylon filters are found suitable forfiltration. The robustness results were represented in table-6, 7, 8, 9 and 10.Table –6: Robustness Parameter Summary table – Flow rate variationFlow Rate Variation (± 0.1 mL/min)Peak Name Low (0.9 mL/min) Actual (1.0 mL/min) High (1.1 mL/min)TF %RSD TF %RSD TF %RSDMetformin 1.2 0.1 1.2 0.2 1.4 0.1Table –7: Robustness Parameter Summary table – Column oven temperature variationColumn oven temperature variation (± 5°C)Peak NameLow (20°C) Actual (25°C) High (30°C)TF %RSD TF %RSD TF %RSDMetformin 1.3 0.0 1.3 0.0 1.4 0.0International Journal of Science Innovations and Discoveries Vol 1, Issue 3, November-December 2011418
Thamma Narendra Kumar et al., IJSID, 2011, 1 (3), 395-421Table –8: Robustness Parameter Summary table – Mobile phase organic composition variationMobile phase organic composition variation (± 10%)Peak NameLow (90%) Actual (100%) High (110%)TF %RSD TF %RSD TF %RSDMetformin 1.3 0.0 1.3 0.0 1.4 0.0Table –9: Robustness Parameter Summary table – Buffer pH variation in mobile phaseBuffer pH variation in mobile phase (± pH 0.10)Peak NameLow (pH 4.90) Actual (pH 5.00) High (pH 5.10)TF %RSD TF %RSD TF %RSDMetformin 1.3 0.1 1.3 0.0 1.3 0.1TF signifies the tailing factor of peak, %RSD signifies the % relative standard deviation of peak response from sixreplicate injectionsTable –10: Filter validation parameter Summary tableFilter validationSimilarity factor for standard solutionSample N° 0.45 µm PVDF Filter 0.45 µm Nylon Filter1 1.00 1.002 1.00 1.00Similarity factor for test solutionSample N° 0.45 µm PVDF Filter 0.45 µm Nylon Filter1 1.00 1.002 1.00 1.00Solution stability:Solution stability in the assay method was carried out by leaving both the test solution of sample andreference standard in tightly capped volumetric flasks at room temperature for 1 day and after 2 days.The % RSD of the assay of actives during solution stability experiments were within 1%. No significantchanges were observed in the content during solution stability experiments. The solution stability experiment dataconfirms that the sample solutions used during assay were stable for 2 days.CONCLUSIONThe simple reverse phase LC method developed for quantitative analysis of Metformin Hydrochloride inpharmaceutical dosage forms is precise, accurate, linear, robust and specific. Satisfactory results were obtainedfrom validation of the method. The method is stability indicating and can be used for routine analysis of productionsamples and stability samples of actives in pharmaceutical dosage forms.REFERENCES1. Lord JM, Flight IHK, Norman RJ. Metformin in polycystic ovary syndrome: systematic review and metaanalysis.BMJ. 2003;327(7421):951–3.2. Marchesini G, Brizi M, Bianchi G, Tomassetti S, Zoli M, Melchionda N. Metformin in non-alcoholicsteatohepatitis.Lancet. 2001;358(9285):893–4. báñez L, Ong K, Valls C, Marcos MV, Dunger DB, de Zegher F.Metformin treatment to prevent early puberty in girls with precocious pubarche. J Clin Endocrinol Metab.2006;91(8):2888–91.International Journal of Science Innovations and Discoveries Vol 1, Issue 3, November-December 2011419
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