Aim: a simple, rapid, precise, accurate and economic stability-indicating RP-HPLC assay method was developed and

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1 Available Online through ISSN: X CODEN: IJPTFI Research Article STABILITY INDICATING VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF METFORMIN HCL AND MIGLITOL IN BULK AND PHARMACEUTICAL DOSAGE FORM SK. Masthanamma*, SK.Naseema, SK.Reehana, SK.Ammaji Department of Pharmaceutical Analysis, University College of pharmaceutical science, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur Andhra Pradesh (India). Received on: Accepted on: Abstract Aim: a simple, rapid, precise, accurate and economic stability-indicating RP-HPLC assay method was developed and validated for simultaneous estimation of Metformin HCl, Miglitol bulk drugs and commercial tablets. Method: The method has shown adequate separation of Metformin HCl and Miglitol from their degradation products. Separation was achieved on a Zorbax C18 (250mm 4.6mm i.d; 5μm) column at wavelength of 270nm, using a mobile phase Orthophosphoric acid (0.1%) ph 2.1 and Methanol (50/50 ) in an isocratic elution mode at a flow rate of 1.2 ml/min. Results: The retention time for Metformin HCl and Miglitol were found to be and 3.894min respectively. The above drug combinations were subjected to acid, base, neutral hydrolysis, thermal and photolytic stress conditions. Thus stressed samples were analyzed by the proposed analytical method. Quantitation was achieved with UV detection at 270 nm based on peak area with linear calibration curves at concentration ranges μg/ml for Metformin HCl and μg/ml for Miglitol (R2 > for both drugs). The LOD s were μg/ml and μg/ml for Metformin HCl, Miglitol respectively. The LOQ s were found to be 0.015μg/ml for MET HCl and μg/ml for MIG. Conclusion: The method was found to be specific and stability indicating as no interfering peaks of degrades and excipients were observed. The proposed method is hence suitable for application in quality-control laboratories for quantitative analysis of both the drugs individually and in combination dosage forms, since it is simple and rapid with good accuracy and precision. Keywords: Forced degradation studies, Metformin HCl, Miglitol, RP-HPLC, Stability indicating assay. IJPT April-2017 Vol. 9 Issue No Page 28590

2 Introduction: SK. Masthanamma*et al. /International Journal of Pharmacy & Technology Metformin [1-2] HCl [fig.1] is chemically 1- carbamimidamido-n, N-dimethylmethanimidamide. With molecular formula C 8 H 17 NO 5 and mg molecular weight. It is most widely prescribed anti-diabetic drug in the world used to treat type 2 diabetes. Metformin helps to control the amount of glucose (sugar) in blood. It decreases the amount of glucose and also increases body's response to insulin, a natural substance that controls the amount of glucose in the blood. It is not used to treat type 1diabetes. It is also used for treatment of gestational diabetes, polycystic ovary syndrome (PCOS). It works by decreasing hyperglycemia primarily by suppressing glucose production by the liver (hepatic gluconeogenesis). It helps to reduce LDL cholesterol and triglyceride levels, and is not associated with weight gain. Metformin comes as a liquid, as a tablet, and as an extended-release (long-acting) tablet taken orally. It is used alone or with other medication. Very rare but serious side effect with Metformin is lactic acidosis. Miglitol [3-4] [fig.2] is chemically (2R, 3R, 4R, 5S)-1-(2-hydroxyethyl)-2-(hydroxymethyl) piperidine-3, 4, 5-triol, with molecular formula C 8 H 17 NO 5. Its molecular weight is mg.Miglitol inhibits glycoside hydrolase enzymes called alpha-glucosidases thereby slowing the appearance of sugar in the blood after meal. It works by slowing down the absorption of carbohydrates from diet, so that blood sugar does not rise as much after meal. Alphaglucosidases inhibitors are used to help control blood sugar levels that are not controlled by diet and exercise alone. It is believed that strict control of blood sugar in people with diabetes decreases the risk of eye, kidney and nerve damage. Objective of study: As literature survey reveals that a number of methods were reported for the estimation of these drugs in bulk and combined dosage form like Simultaneous estimation of Metformin HCl and Miglitol in tablet dosage form by RP-HPLC [8-23], UV-spectrophotometric method [6-7]. A number of spectrophotometric and chromatographic analytical methods were reported for the estimation of these drugs in their formulations. However, these methods lack stability indicating nature. Also, none of the reported procedures enable analysis of both the drugs in pharmaceutical dosage forms in presence of their degradation products. In the present investigation, IJPT April-2017 Vol. 9 Issue No Page 28591

3 an attempt was made to develop a simple, rapid, precise and accurate stability indicating RP-HPLC assay method for simultaneous estimation of Metformin HCl and Miglitol presence of their degradation products This proposed method can be successfully employed for quality control during manufacture and for assessment of the stability of both drugs in bulk samples and combined dosage forms. Materials and Methods: Drug substance: Metformin HCl and Miglitol (working standard 99.12% and 99.73%) were obtained as gift sample from Rainbow pharma training lab, Hyderabad, India. Pharmaceutical tablet formulation of MIGNAR25-MFwas purchased from local pharmacy. Methanol (HPLC Grade, MERCK), Orthophosphoric acid (HPLC grade, MERCK), Hydrochloric acid (AR grade), sodium hydroxide, hydrogen peroxide (AR grade) and HPLC grade water were used for the entrained study. Instrumentation: All HPLC experiments were carried out on a Waters Alliance 2695 separation module, with waters 2996 photodiode array detector in isocratic mode using Auto sampler. Data collection and processing was done using EMPOWER PDA 2 software. The analytical column used for the separation was Zorbax C18, mm I.D., 5 μm particle size, Other equipments used were ultra-sonicator (model 3210, Branson Ultrasonic s Corporation, Connecticut, USA), Analytical balance (contech balance). Preparation of solutions Diluents: Diluents was prepared by mixing solvent A and solvent B (a: b, 50/50) Preparation of 0.1M Orthophosphoric acid buffer solution: Prepared by dissolve 1ml of OPA with 1000ml of HPLC grade water. Mobile phase: Mobile phase was prepared by mixing OPA (0.1%, ph-2.1,) and Methanol (50/50). It was filter to 0.45u membrane filter to remove the impurities otherwise they may interfere in the final chromatogram and it was sonicated for 15min to remove the undissovable gases and air bubbles. Preparation of 0.1N HCL 0.1 N HCL was prepared by taking 0.08ml of conc. HCL in 100ml of HPLC grade water. Preparation of 0.1N NaoH 0.1N NaoH was prepared by taking 0.4mg of NaoH in 100ml of HPLC grade water. Preparation of Hydrogen peroxide (3%) Hydrogen peroxide was prepared by taking 3ml of hydrogen peroxide in 100ml of HPLC grade water. IJPT April-2017 Vol. 9 Issue No Page 28592

4 Standard solution SK. Masthanamma*et al. /International Journal of Pharmacy & Technology Standard solutions of Metformin HCl and Miglitol were prepared by dissolving 500mg of Metformin HCl and 25mg of Miglitol in two separate 50ml volumetric flasks contain 10ml of HPLC grade water in each flask, sonicate for 5min and final volume were made up to the mark with HPLC grade water. From these stock solutions take 2.5ml from each flask and transfer into two separate 25ml volumetric flasks, the final volumes were made up to the mark with HPLC grade water to get the concentrations of 1005µg/ml of Metformin HCl and 0.55µg/ml Miglitol respectively. Chromatographic conditions: The mobile phase was a mixture of OPA (0.1%, ph 2.1) and Methanol (50/50 v/v). The contents of the mobile phase were filtered, before it was used, through 0.45 μm membrane filter, degassed with a helium spurge for 15 min and pumped from the respective solvent reservoirs to the column at a flow rate of 1.2 ml/min, Zorbax C18 (250*4.6mm I.D, 5μm. The column temperature was maintained at 30 o c and run time 20mins. The injection volume of samples was 10μL. The analyte was monitored at 270nm. The chromatographic conditions were shown in Table.1.and obtained chromatogram shown in fig.3. Figure.3. Optimised chromatography. Method Development: To saturate the column, the mobile phase was pumped for about 30 minutes thereby to get the base line corrected. After no. of trials optimum chromatographic conditions were fixed for better separation. The separate standard calibration lines were constructed for each drug. A series of aliquots were prepared from the above stock solutions using HPLC grade water to get the concentrations µg/ml Metformin HCl and µg/ml Miglitol. Each concentration 6 times was injected into chromatographic system. Each time peak area and retention time recorded separately for both the drugs. Calibration curves were constructed by taking average peak area on y-axis and concentration on x-axis separately for both the drugs. From the calibration curves regression equations were calculated as shown in the figure no.4 & 5. IJPT April-2017 Vol. 9 Issue No Page 28593

5 Area SK. Masthanamma*et al. /International Journal of Pharmacy & Technology y = 16039x R² = Ряд1 Линейная (Ряд1) Fig.4. Calibration curve of Metformine HCl y = x R² = Ряд1 Линейная (Ряд1) Concentration (µg/ml) Fig.5. Calibration curve of Miglitol Estimation of Metformin HCL and Miglitol in Tablet Dosage Form: For the analysis of drugs, 20 tablets were weighed and triturated in a glass mortar and quantity of powder equivalent to 500mg of Metformin HCl was transferred to 50ml volumetric flask and dissolved in sufficient quantity of HPLC grade water. It was sonicated for 5mins and volume was made up to 50ml HPLC grade water. It was filter through 0.45μ membrane filter. From this solution transfer 2.5ml into 25ml volumetric flasks, the final volume were made up to the mark with HPLC grade water to get the concentrations of 100μg/ml of Metformin HCl and 5μg/ ml of Miglitol respectively. The test concentration is injected 6 times in to chromatographic system. Each time peak area and retention time was recorded and the results obtain were as shown in the table no.2. Table.2: Results of Marketed Formulation analysis: Drug name Lablled claim(mg) Test concentratio n(µg/ml) Meam Amt found (µg/ml) (n=6) % Estimated Amt Metformin HCl Miglitol Method validations: The analytical method was validated for various parameters as per ICH guidelines. IJPT April-2017 Vol. 9 Issue No Page 28594

6 Linearity SK. Masthanamma*et al. /International Journal of Pharmacy & Technology The linearity of the method was determined in concentration range of μg/ml for MET HCl and μg/ml for MIG. Each solution was injected in triplicate. The average peak area versus concentration data of both drugs was treated by least squares linear regression analysis. Linearity was checked over the same concentration range on three consecutive days and the results obtained from as shown in tableno.3. Table.3: Linearity studies results Parameters MET HCl MIG Linearity range (µg/ml) Regression line equation Correlation coefficient (r) No of data points LOD (µg/ml) LOQ (µg/ml),%rsd y = 16039x , y =385172x ,0.35 Specificity and Selectivity Specificity is the degree to which the procedure applies to a single analyte and is checked in each analysis by examining blank matrix samples for any interfering peaks. The specificity of the method was evaluated with regard to interference due to presence of any other excipients. Two different samples were injected and studied with respective excipients. The HPLC chromatograms recorded for the drug matrix (mixture of the drug and excipients) showed almost no interfering peaks with in retention time ranges. Fig. 6 & 7 show the respective chromatograms for MET HCl and MIG with Blank and Placebo. The figures shows that the selected drugs were cleanly separated. Thus, the HPLC method proposed in this study was selective. (a) (b) Fig.6 (a). Specificity chromatogram with Blank (b) Specificity chromatogram with Placebo IJPT April-2017 Vol. 9 Issue No Page 28595

7 a b c d e Fig.7 (a-e): a) Acid Degraded sample b) Base Degraded sample c) Thermal degraded sample d) Peroxide degraded sample e) Photo degradation sample. Accuracy, as Recovery Accuracy was evaluated in triplicate, at three different concentrations equivalent to 50, 100, and 150% of the target concentration of active ingredient, by adding a known amount of each of the Standard to a sample of known concentration of both drugs and calculating the % of recovery, and the results obtained from as shown in table no.4. IJPT April-2017 Vol. 9 Issue No Page 28596

8 Table.4: Accuracy studies results SK. Masthanamma*et al. /International Journal of Pharmacy & Technology Drug Pre analysed Recovery Amt of drug Amt of %recovery Acceptece name concentratio level added(µg/ml) drug criteria n taken found (µg/ml) (µg/ml)n =3 Metformin HCl 50 Miglitol % % % % Precision: Precision is the degree of repeatability of an analytical method under normal operational conditions. Precision of the method was determined with the standard and test sample. The precision of the method was verified by repeatability and the intermediate precision studies. Method repeatability was achieved by repeating the same procedure of preparation of solution six times and injecting. Intermediate precision was performed by performing the same procedure on the same day for intra-day precision. The inter day precision of the method was checked by performing same procedure on different days under same experimental conditions. The repeatability of sample application and measurement of peak area were expressed in terms of relative standard deviation (%RSD) and results obtained were as shown in table no.5. Table.5: Precision studies results. Day of analysis %Recovery±SD; %RSD Intraday Precision Metformin HCl (µg/ml) Day ± ± ± Day ± ± ±0.03 Day ± ± ±0.33 Miglitol (µg/ml) Day ± ± ±0.31 IJPT April-2017 Vol. 9 Issue No Page 28597

9 LOD and LOQ SK. Masthanamma*et al. /International Journal of Pharmacy & Technology Day1 99.0± ± ± Day ± ± ±0.18 Interday Precision HYD HCl (µg/ml) Day0,1, ± ± ± ISD (µg/ml) Day0,1,2 99.0± ± ± The Limit of Detection (LOD) and Quantitation (LOQ) for both MET HCl and MIG were determined using standard deviation method according to ICH guidelines Q2 (R1). LOD is lowest amount of analyte in a sample that can be detected but not necessarily quantities as an exact value under the stated experimental conditions. The detection limit is usually expressed as the concentration of analyte. The standard deviation and response of the slope LOD = 3.3*standard deviation (ϭ)/s The LOQ of an analytical procedure is the lowest amount of an analyte of a sample which can be quantitatively determined with suitable precision and accuracy. The standard deviation and response of the slope and the results obtained were as shown the table no.6 Table.6: LOD and LOQ results Robustness: Drug name Area Retention time LOD LOQ Metformine HCl Miglitol LOQ= 10* standard deviation (ϭ)/s To evaluate the robustness of the method, the chromatographic conditions were deliberately Altered and degree of reproducibility was evaluated. During robustness testing each condition was varied separately, all other conditions being held constant at the optimized values. Robustness of the proposed method was assessed with respect to small alterations in the flow rate (1.2± 0.1ml/min), and Temperature (30 0 c ± 2 0 c) and the results obtained were as shown the table no.7. IJPT April-2017 Vol. 9 Issue No Page 28598

10 Table.7: Robustness studies results SK. Masthanamma*et al. /International Journal of Pharmacy & Technology Method Conditios Retention Time(R t) Area %Recovery Parameters MET HCl MIG MET HCl MIG MET HCl MIG Temp Temp Temp Flow Flow Flow System suitability parameters For assessing system suitability, six replicates of working standards samples of MET HCl and MIG were injected and studied the parameters like plate number(n), tailing factor(k),resolution, relative retention time and peak asymmetry of samples. The results were show in tableno.8. Table.8: System suitability parameters of MET HCl and MIG. Values obtained (n=6) Parameters MET HCL MIG Plate count Tailing Factor R t (min) resolution Asymmetry Degradation sample Preparation Weigh accurately 20tablets and crush into fine powder from this take 100mg (equivalent to 50mg MET HCl 2.5mg MIG) of powdered sample into a 100ml volumetric flask dissolve and dilute to volume with HPLC grade water and filter the solution using 0.45µ Nylon filter. Acid hydrolysis Transfer 1ml (500ug/ml MET HCl) of above stock solution to10ml volumetric flask and add 1ml of 0.1N HCL and reflux for 30min at 60 0 c.cool to room temperature and neutralize with 1ml of 0.1N NaoH and makeup volume with HPLC grade water. IJPT April-2017 Vol. 9 Issue No Page 28599

11 Base Hydrolysis SK. Masthanamma*et al. /International Journal of Pharmacy & Technology Transfer 1ml (500ug/ml MET HCl) of above stock solution to10ml volumetric flask and add 1ml of 0.1N NaoH and reflux for 30min at 60 0 c.cool to room temperature and neutralize with 1ml of 0.1N HCl and makeup volume with HPLC grade water. Peroxide hydrolysis Transfer 1ml (500ug/ml of MET HCl) of above stock solution to10ml volumetric flask and add 1ml of 3%v/v of H and reflux for 30min at 60 0 c.cool to room temperature and makeup volume with HPLC grade water. Thermal Degradation Weigh accurately 20tablets and crush into fine powder and transfer powder to 200mg (equivalent to 100mg MET HCl and 5mg MIG) powder into petridish. Heat the sample in oven for about 6hrs at c. From this weigh accurately 100 mg of powdered sample into a 100ml volumetric flask dissolve and dilute to volume with HPLC grade water. Transfer 1ml of above stock solution to10ml volumetric flask and filter the solution using 0.45µ Nylon filter. Photolytic Degradation Photolytic degradation study was carried out by exposing the accurately weighed 200mg (equivalent to 100mg MET HCl and 5mg MIG) of tablet powder to UV light in a photolytic chamber at 2600 lux for 24 hr, After 24hrs weigh accurately 100 mg of powdered sample into a 100ml volumetric flask dissolve and dilute to volume with HPLC grade water.transfer 1ml of above stock solution to10ml volumetric flask and filter the solution using 0.45µ Nylon filter. Using the peak purity test, the purity of the drugs peaks were checked at every stage of above-mentioned studies. Forced Degradation Studies Stress-degradation studies of the drug substances can help identifying the possible degradation products which can in turn help establishing the degradation pathways and the intrinsic stability of the molecule and validate the stabilityindicating power of the analytical procedures used. The chromatograms of Metformin HCl and Miglitol, after being subjected to different mild and drastic degradation conditions, were compared with blank solutions injected in a similar manner and with recently prepared solutions. These results showed the specificity of the developed method clearly. The results indicated that in the stressed degradation studies using the optimized methods, degradation peaks for MET HCl and MIG did not affect the drug peak. The mass balance of Metformin HCl and Miglitol under each stress condition was found 100% and, moreover, assay of each unaffected compound in the tablets confirmed the stability indicating nature of the method. MIG was relatively more labile than MET HCl in photolytic and thermal IJPT April-2017 Vol. 9 Issue No Page 28600

12 degradative conditions while MIG was more susceptible than MET HCl in acidic, basic, neutral and oxidative. The results from forced degradation studies were summarized in Table no.9.representative Chromatograms obtained from forced degradation studies are shown in Fig.7a-e). Table.9: Forced Degradation Studies Stress conditions Metformin HCl Miglitol % degradation % degradation Acidic/0.1 M HCl/60 C reflux/48 h Basic/0.1 M NaOH/60 C reflux/48 h Oxidizing/3% H2O2/cool at RT/30min Thermal/105 C/6hr Photolysis/UV light The degradation behavior and degradation products of MET HCL and MIG were found to be similar in combination drug product and in bulk drugs under various stress conditions assessed. The study was not intended to identify degradation products but merely to show they would not interfere if and when present. To conclude, the results of stress testing studies indicate a high degree of specificity of this method for both MET HC land MIG. Solution stability The stock solution showed no significant change in analyte composition, retention time and peak areas of MET HCl and MIG after 1 weeks of storage at room temperature. This was sufficient for the whole analytical process Results and Discussion Optimized chromatographic conditions: Most of all reported HPLC methods till date use C-8 or C-18 columns. Most of these uses Complex mobile phase compositions. Hence, attempts were directed towards development of a Simple and better method on commonly used C18 column with good resolution. Different logical modifications were tried to get good separation among the drugs and the degraded products. These changes included change in mobile phase composition in isocratic elution as well as gradient modes on different C18 columns. The optimized chromatographic conditions (fig.3).the best peak shape and maximum separation was achieved with mobile phase consist of 0.1%OPA (ph 2.1) and methanol (a: b, 50/50). Peak symmetry and reproducibility were obtained on Zorbax C18, 250mm 4.6mm I.D; 5μm).The detection wavelength at 270nm, a flow rate of 1.2ml/min yielded optimum separation and peak symmetry as shown table 1. IJPT April-2017 Vol. 9 Issue No Page 28601

13 Table.1: Optimized Chromatographic conditions: Column Zorbax 250*4.6mm,5µm Flow rate 1.2ml/min Wavelength 270nm Column temperature 30 o c Injection volume 10µl Run time 20min Diluents HPLC grade water Elution Isocratic Mobile phase 0.1%OPA (ph 2.1),Methanol(50/50 v/v) Degradation studies Results are tabulated in table no.9. Acid hydrolysis (figure 7 a) Upon performance of acid degradation studies 98% of Metformin HCl and 94% of Miglitol was degraded. Base hydrolysis (fig.7b) Upon performance of base degradation studies 96% of Metformin HCl and 93% of Miglitol was degraded. Peroxide hydrolysis (fig.7c) Upon performance of peroxide degradation studies 98% of Metformin HCl and 94% of Miglitol was degraded. Thermal degradation (fig.7d) Upon performance of Thermal degradation studies 97% of Metformin HCl and 98% of Miglitol was degraded. Photolytic degradation (fig.7e) Upon performance of Photolytic degradation studies 97% of Metformin HCl and 98% of Miglitol was degraded. Linearity, LOD and LOQ The calibration plot was linear over the concentration range investigated (50-150μg/ml; n = 3) and ( μg/ml; n = 3) for MET HCl and MIG respectively (figure 7, 8). Average correlation co-efficient r= for both drug candidates with %RSD values 2.0 across the concentration ranges studied, was obtained from regression analysis. The LOD for MET HCl and MIG were found to be 0.005μg/ml and μg/ml respectively. The LOQ that produced the requisite precision and accuracy was found to be 0.015μg/ml for MET HCl and μg/ml for MIG. The resultant %RSD values were 1.00 %( table no.5).the regression results indicate that method was linear in the IJPT April-2017 Vol. 9 Issue No Page 28602

14 concentration range studied (table.5) and can be used for detection and quantification of MET HCl and MIG in a very wide concentration range. Accuracy and Precision Accuracy as recovery was evaluated by spiking previously analyzed test solution with additional Standard drug at three different concentration levels (table-3). Recovery of standard drugs added was found to be 99.31% for Metformin HCl and 97.19% for Miglitol with the value of RSD less than 1% indicating that the proposed method is accurate for the simultaneous estimation of both drugs from their combination drug products in presence of their degradation products. The low RSD values indicate the repeatability and reproducibility of the Method (table 4). Robustness Results of the robustness (table.7) study are depicted in Table no.3. The elution order and resolution for both components were not significantly affected. RSD of peak areas were found to be well within the limit of 2.0%. Discussion A simple, rapid, accurate and precise stability-indicating HPLC analytical method has been developed and validated for the routine quantitative analysis of Metformin HCl and Miglitol in API and combined dosage forms. The results of stress testing undertaken according to the ICH guidelines reveal that the method is specific and stability-indicating. The proposed method has the ability to separate these drugs from their degradation products in tablet dosage forms and hence can be applied to the analysis of routine quality control samples and samples obtained from stability studies. Conclusion A simple, rapid, accurate and precise stability-indicating HPLC analytical method has been developed and validated for the quantitative analysis of Metformin HCl and Miglitol in bulk drugs and combined dosage forms. Stress testing (or forced degradation studies) is an important part of drug development process and the pharmaceutical industry has much interest in this area. The results of stress testing undertaken according to the ICH guidelines reveal that the method is specific and stability-indicating. The proposed method has the ability to separate these drugs from their degradation products in tablet dosage forms and hence can be applied to the analysis of routine quality control samples and samples obtained from stability studies. Acknowledgment: Authors are very thankful to principal, University College of pharmaceutical sciences, Acharya Nagarjuna University, Guntur, for providing the library facilities for literature survey to carryout entire study. IJPT April-2017 Vol. 9 Issue No Page 28603

15 Authors also thankful to Rainbow pharma training lab, Hyderabad, India, for providing Metformin HCl and Miglitol working standard as gift sample and HPLC Instrument to carry out Research work. References 1. Available from: Metformin. 2. Available from: 3. Available from: Miglitol. 4. Available from: 5. Arayne MS, Sultana N, Zuberi MH, Siddiqui FA. Spectrophotometric Quantitation of Metformin in Bulk Drug and Pharmaceutical Formulations using Multivariate Technique. Indian journal of pharmaceutical sciences 2009; 71(3): Narasimha RD, Prasada RM, Hussain J, Sumanoja S, Rajeswara RV. Naga Lakshmi. Method development and validation of forced degradation studies of metformin hydrochloride by using uv spectroscopy. IJPCBS 2013; 3: AbuRuza S, Millershipb BJ, McElnayb J, Chromatography B. The development and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Journal of chromatography.2005; 817(2): Shrivastava B, Stability-Indicating RP-HPLC Method for Estimation of Miglitol in Bulk and Tablets. Indian journal of pharmaceutical sciences. 2010; 72(6): Chungath T, Padmanabha RY, Devanna N. Thomas Development of validated RP-HPLC method for the estimation of Miglitol in tablet formulations. Research Journal of Pharmaceutical Biological and Chemical Sciences 2011; 2(3): Li X, Wang Y, Wang J, Fawcett JP, Zhao L, Gu J. Determination of Miglitol in human plasma by liquid chromatography/tandem Mass spectrometry. Rapid communications in mass spectrometry: RCM 2007; 21(2): Ibrahim FA, Ali FA, Ahmed SM, Tolba MM. Kinetic determination of acarbose and Miglitol in bulk and pharmaceutical formulations using alkaline potassium permanganate. International journal of biomedical science: IJBS 2007; 3(1): IJPT April-2017 Vol. 9 Issue No Page 28604

16 12. Sejal RP, Prachi VK, Ritu VK, Ravikiran M, Nargun LVG. Development and Validation of Analytical Method for Quantitative Estimation of Miglitol and Metformin in Combined Dosage Form. Journal of Applied Pharmaceutical Science 2012; 2(6): Arayne MS, Sultana N, Zuberi MH. Development and validation of RP-HPLC method for the analysis of metformin. Pakistan journal of pharmaceutical sciences 2006; 19(3): Madhukar A, Prince A, Vijaykumar R, Sanjeev Y, Raghuprata D, Jagadeeswar.K, Simple and Sensitive analytical method development and validation of Metformin hydrochloride by RP-Hplc. Journal of Pharmacy and Pharmaceutical Sciences. Journal of chromatographic science 2011; 3(3): Narendra KT, Mohan RK, Sreenivasulu K, Novel RP, Raju R, V. NS, et al. Chandra for the Estimation of Metformin hydrochloride in pharmaceutical dosage forms International Journal of Science Innovations and Discoveries. Journal of chromatographic science 2011; 1(3): Yunbiao W, Fawcettb, Yingwu Jingkai Gua, Paul Xu Bai. Rapid and sensitive liquid chromatography tandem mass spectrometric method for the Quantitation of metformin in human plasma Journal of chromatography B 2004; 808(2 ): Mousumi K, Choudhury PK, Indian J. HPLC method for estimation of metformin hydrochloride in formulated microspheres and tablet dosage form Pharm. Sci 2009;71(3): Sultana N, Arayne MS, Shafi N, Siddiqui FA, Hussain A. Development and validation of new assay method for the simultaneous analysis of diltiazem, metformin, pioglitazone and rosiglitazone by RP-HPLC and its applications in pharmaceuticals and human serum. Journal of chromatographic science 2011; 49(10): Narsimha RD, Chandana M, Hplc, Pioglitazone H. Method Development and Validation of RP-HPLC for Simultaneous Analysis of Three Component Tablet Formulation containing Metformin and Glibenclamide. International Journal of PharmTech Research 2012; 4: Florentin T, Monica A. specificity of an analytical hplc assay method of metformin hydrochloride Revue Roumaine de Chimie. Journal of chromatographic science 2007; 52(6): Mastanamma.SK1*, G. Rambabu, G, Saidulu. P and Tejaswini. I. S., Designing of forced degradation studies and development of validated stability indicating method for simultaneous estimation of desloratadine and montelukast odium in their formulation, 7, 2015, IJPT April-2017 Vol. 9 Issue No Page 28605

17 22. Masthanamma Sk*, Anil Kumar T, Bhagya Srivani G, Anantha Sridhar T, Siva Sankar Naik B, Vinay Kumar V, Stability-Indicating Validated Reversed Phase-High Performance Liquid Chromatography method for Simultaneous determination of Cobicistat And Atazanavir Sulfate in bulk And Pharmaceutical dosage form,9, 2016, Saidulu. P*, Sk. Mastanamma.sk, Stability Indicating Gradient RP-HPLC Method for the Simultaneous Estimation of Lamivudine, Abacavir and Dolutegravir in Bulk and their Combined Dosage Form, International Journal of Pharmaceutical Sciences Review and Research,37, 2016, IJPT April-2017 Vol. 9 Issue No Page 28606

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