Method Development & Validation of Metformin & Emphagliflozine

MethodDevelopment & Validation of Metformin & Emphagliflozine dosage forms byRPHPLC Student: B.Jaffar Hussain Reg.No14Q21S0702Guige G.Somasekhar CHAPTER-1INTRODUCTION Pharmaceuticalanalysis simply means analysis of pharmaceuticals. Webster’ dictionary definesa pharmaceutical is a medical drug.

A more appropriate term for apharmaceutical is active pharmaceutical ingredient (API) or active ingredientto distinguish it from a formulated product or drug product is prepared byformulating a drug substance with inert ingredient (excipient) to prepare adrug product that is suitable for administration to patients. Research anddevelopment (R) play a very comprehensive role in new drug developmentand follow up activities to ensure that a new drug product meets theestablished standards is stable and continue to approved by regulatoryauthorities ,assuring that all batches of drug product are made to the specificstandards utilization of approved ingredients and production method becomes theresponsibility of pharmaceutical analysts in the quality control (QC) orquality assurance department . The methods are generally developed in ananalytical R department and transferred to QC or other departments asneeded. At times they are transferred to other divisions.By now it shouldbe quite apparent that pharmaceutical analysts play a major role in assuringthe identity, safety, efficacy, and quality of drug product, safety andefficacy studies required that drug substance and drug product meet two criticalrequirements.

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1.                 Established identity and purity.2.                 Established bioavailability/dissolution.   Chapter 2: METHOD DEVELOPMENT ANDVALIDATION2.1 Introduction to Method Development            The numberof drugs introduced into the market is increasing every year.

These drugs maybe either new entities or partial structural modification of the existing one.Often a time lag exists from the date of introduction of a drug into the marketto the date of its inclusion in pharmacopoeias. This happens because of thepossible uncertainties in the continuous and wider usage of these drugs,reports of new toxicities (resulting in their withdrawal from the market),development of patient resistance and introduction of better drugs bycompetitors. Under these conditions, standards and analytical procedures forthese drugs may not be available in the pharmacopoeias. It becomes necessary,therefore to develop newer analytical methods for such drugs. 11,12                Analytical methods should be usedwithin good manufacturing practice (GMP) and good laboratory practice (GLP)environments, and must be developed using the protocols set out in theInternational Conference on Harmonization (ICH) guidelines (Q2A and Q2B).

13,14            Chapter:3 DRUG PROFILE             Empagliflozin     Empagliflozinis a sodium glucose co-transporter-2 (SGLT-2) inhibitorindicated as an adjunct to diet and exercise to improve glycemic control inadult patients with type 2 diabetes. SGLT2 co-transporters are responsible forreabsorption of glucose from the glomerular filtrate in the kidney. Theglucuretic effect resulting from SGLT2 inhibition reduces renal absorption andlowers the renal threshold for glucose, therefore resulting in increasedglucose excretion. Additionally, it contributes to reduced hyperglycaemia andalso assists weight loss and blood pressure reduction.Categories:·        DrugsUsed in Diabetes·        AlimentaryTract and Metabolism·        BloodGlucose Lowering Drugs, Excl.

InsulinsWeight:  450.91Chemical Formula:C23H27ClO7IUPAC Name:(2S,3R,4R,5S,6R)-2-4-chloro-3-({4-(3S)-oxolan-3-yloxyphenyl}methyl)phenyl-6-(hydroxymethyl)oxane-3,4,5-triol  Chapter:4  REVIEW OF LITERATURE ·        K.S.

LAKSHMI et.al.. Asimple, sensitive and rapid reverse phase high performance liquidchromatographic method was developed for the estimation of Metformin Hcl (MET)and Pioglitazone (PIO) in pure and in pharmaceutical dosage forms. A Gemini C18column (150×4.6mm, 5µ) was used with a mobile phase containing a mixture ofAcetonitrile and Ammonium Acetate buffer (pH-3) in the ratio of 42: 58. Theflow rate was 0.

3ml/min and effluents were monitored at 255nm and eluted at5.17min (MET) and 8.1min (PIO). Calibration curve was plotted with a range from0.5-50 µg/ml for MET and 0.3-30 µg/ml for PIO. The assay was validated for theparameters like accuracy, precision, robustness and system suitabilityparameters.

The proposed method can be useful in the routine analysis for thedetermination on metformin and pioglitazone in pharmaceutical dosage forms.·        Manasaet al.. In the present study, two analytical methods were developed forthe estimation of Dapagliflozin in API.

Method A: RPHPLC method, Method B: UVspectroscopic method. In method A, the drug showed linearity in the range of25-150µg/ml with a correlation coefficient (r2 ) of 0.999, where as in methodB, the linearity range was found to be 1-5µg/ml with a correlation coefficientof (r2 ) 0.999. Both the methods were validated for different validationparameters such as linearity, accuracy, precision, detection limit,quantitation limit, robustness and ruggedness and the results were found to bewithin the acceptance limits as per the guidelines of International Conferenceon Harmonization (ICH).·        Kavitha.

K. Y et al.. A simple, RP-HPLC method was established fordetermining linagliptin and metformin in pharmaceutical formulations.

Linagliptin , metformin and their degradation products were separated using C8column with Acetonitrile: Water: Methanol (25:50:25 (v/v/v) to pH 4.1 with 0.1%orthophosphoric acid as the mobile phase. Detection was performed at 243 nmusing a diode array detector. The method was validated using ICH guidelines andwas linear in the range 5-30µg/ and 10-100 µg /ml for linagliptin and metforminrespectivily. Good separation of both the analytes and their degradationproducts was achieved using this method. The developed method can be appliedsuccessfully for the determination of linagliptin and metformin.     Chapter: 5 NEED FOR THE STUDY5.

0  Analytical Method Development for Pharmaceutical FormulationsQuality investigation plays a very important role inquality specification establishment of chemical drugs. The number of drugs introduced into the market every year .veryoften there is a time lag from the date of introduction of a drug into themarket to the date of its inclusion in pharmacopoeias. Hence, standards andanalytical procedures for these drugs may not be available in thepharmacopoeias. It becomes necessary, therefore to develop newer analyticalmethods for such drugs. Basic criteriafor new method development of drug analysis:·        The drug or drug combination maynot be official in any pharmacopoeias.

·        A proper analytical procedure forthe drug may not be available in the literature due to patent regulations.·        Analytical methods may not beavailable for the drug in the form of a formulation due to the interferencecaused by the formulation excipients.·        Analytical methods for a drug incombination with other drugs may not be available.·        The existing analytical proceduresmay require expensive reagents and solvents.

It may also involve cumbersomeextraction and separation procedures and these may not be reliable.Analytical method development provides the support totrack the quality of the product from batch to batch.  Chapter: 6 AIM ANDPLAN OF WORK  6.0   AIM To develop new RP HPLC method for thesimultaneous estimation of Metformin and empagliflozin  pharmaceutical dosage form. 6.

1   PLAN OF WORK  •        Solubility determination of Metformin andempagliflozin various solvents and buffers.  •        Determine the absorption maxima of both thedrugs in UV–Visible region in different solvents/buffers and selecting thesolvents for HPLC method development.  •        Optimize the mobile phase and flow rates forproper resolution and retention times.

 •        Validate the developed method as per ICHguidelines.   Chapter: 7MATERIALS AND METHODS7.4. Mobile Phase:A mixture of 50 volumesof methanol and 50 volumes of  phosphatebuffer were prepared. The mobile phase was sonicated for 10min to remove gasesand filtered through 0.

45µ membrane filter for degassing of mobile phase. 7.4.

1. Preparation of Mixed PhosphateBuffer:1.625 gm ofpotassium di hydrogenphosphate (KH2PO4)and 0.

3 gm of Dipotassium hydrogen phosphate was weighed anddissolved in 100ml of water and volume was made up to 550ml with water. Adjustthe pH to 4.0 using ortho phosphoric acid. The buffer was filtered through0.45µ filters to remove all fine particles and gases.   Chapter:8.0 RESULTS AND DISCUSSION 8.1.

Solubility StudiesThesestudies are carried out at 25 0 CMETFORMIN:soluble in methanol and in water, very slightly soluble inphosphate buffer.EMPAGLIFLOZIN:Freely soluble in water,soluble in acetonitrile,spraingly soluble in methanol.8.2. DeterminationOf  Working Wavelength (?max)In simultaneous estimation of  two drugs isobestic wavelength is used.Isobestic point is the wavelength where the molar absorptivity is the same fortwo substances that are interconvertible. So this wavelength is used insimultaneous estimation to estimate both drugs accurately.

8.2.1. Preparation of standardstock solution of METFORMIN10mg of METFORMINwasweighed and transferred in to 100ml volumetric flask and dissolved in methanoland then make up to the mark with methanol and prepare 10 µg /ml of solution bydiluting 1ml to 10ml with methanol.8.

2.2. Preparation of standardstock solution of EMPAGLIFLOZIN 10mg of EMPAGLIFLOZINwas weighed in to 100mlvolumetric flask and dissolved in Methanol and then dilute up to the mark withmethanol and prepare 10 µg /ml of solution by diluting 1ml to 10ml withmethanol.8.2.3. Results           The wavelength of maximum absorption(?max) of the drug, 10 ?g/ml solution of the drugs in methanol werescanned using UV-Visible spectrophotometer within the wavelength region of200–400 nm against methanol as blank.

The resulting spectra are shown in thefig. no. 8.1, 8.

2 and 8.3 and the absorption curve shows characteristicabsorption maxima at 240 nm for METFORMIN, 229 nm for EMPAGLIFLOZIN and255nm for the combination. Chapter: 9.0 VALIDATION 9.1 Systemsuitability Standard solutions wereprepared as per the test method and injected into the chromatographic system.

The system suitability parameters like theoretical plates, resolution andasymmetric factor were evaluated. 1.   The % RSD for the retention times of METFORMIN and EMPAGLIFLOZIN Peaksfrom 6 replicate   injections of eachStandard solution should be not more than 2.0 %2.         The % RSD for the peak area responses ofMETFORMIN and EMPAGLIFLOZIN peaks from 6 replicate injections of each standardsolution should be not more than 2.

0%.  3.   The number of theoretical plates (N) for the METFORMINand EMPAGLIFLOZIN peaks is not less than2000.4.  The Tailing factor (T) for the METFORMINand EMPAGLIFLOZIN peak is not more than 2.0.

  Observation    The % RSD for theretention times and peak area of METFORMIN and EMPAGLIFLOZIN were found to beless than 2%. The plate count andtailing factor results were found to be satisfactory and are found to be withinthe limit.    Chapter: 10 Discussion                  A simple and selective LCmethod is described for the determination of Metformin andempagliflozin in tablet dosage forms. Chromatographic separation was achieved ona c18 column using mobile phase consisting of a mixture of 50volumes of methanol and 50 volumes of phosphate buffer with detection of 255 nm. Linearity wasobserved in the range 60-140 µg /ml for Metformin (r2=0.997) and 3-7µg /ml for empagliflozin (r2=0.995) for the amount of drugs estimated by the proposed methods was in goodagreement with the label claim.

   Chapter:11 Conclusion From theabove experimental results and parameters it was concluded that, this newlydeveloped method for the simultaneous estimation Metformin andempagliflozindrugs was found to be simple, precise, accurate andhigh resolution and shorter retention time makes this method more acceptableand cost effective and it can be effectively applied for routine analysis inresearch institutions, quality control department in meant in industries,approved testing laboratories studies in near future.  

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