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METHOD DEVELOPMENT AND VALIDATION OF GLC FOR PHARMACEUTICALS:

METHOD DEVELOPMENT AND VALIDATION OF GLC FOR PHARMACEUTICALS PRESENTED BY; PRESENTED BY: G.PRASAD 14004P1036 M.PHARMACY(PHARMAEUTICAL ANALYSIS) UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES

CONTENTS:

CONTENTS INTRODUCTION INSTRUMENTATION GC DERIVITIZATION DEVELOPING SEPARATION METHODS VALIDATION REFERENCES

INTRODUCTION:

INTRODUCTION Gas chromatography(GC), is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. In gas chromatography, the  mobile phase   is a carrier  gas , usually an  inert  gas such as  helium  or an  unreactive  gas such as  nitrogen . The stationary phase  is a microscopic layeof   liquid  or  polymer  on an inert  solid support , inside a piece of  glass  or  metal  tubing called a column. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time

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INSTRUMENTATION Tank of carrier gas Flow regulator and flow meter Injection port coloumn Temperature controlled device Detector Recorder

DETECTORS:

DETECTORS The three most common detectors in GC are Thermal conductivity detector(TCD) Flame ionization detector(FID) Electron capture detector(ECD) Thermal conductivity detector : It measure heat conductivity which is different for different gases.

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FID and ECD types respond to changes in electron currents, the electrons are produced in a flame by burning the sample, or by exposing the sample to a radioactive source. FID : flame ignites and ionizes the combusible sample components as the carrier gas passes in to it, after which the ions are collected at the electrodes producing current. ECD : The carrier gas is passed through a cell containing a beta source , which ionize the carrier gas.

GC Derivatization:

GC Derivatization Derivatization is the process of chemically modifying a compound to produce a new compound which has properties that are suitable for analysis using a GC. To permit analysis of compounds not directly amenable to analysis due to, for example, inadequate volatility or stability. Improve chromatographic behaviour or detectability . Increases volatility (i.e. sugars): – Eliminates the presence of polar OH, NH, & SH groups – Derivatization targets O,S, N and P functional groups (with hydrogens available

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Types of Derivatization: Silylation Alkylation Acylation Silylation: Silylating reagents react with compounds containing active hydrogen's Silylation produces silyl derivatives which are more volatile, less stable, and more thermally stable. Replaces active hydrogen's with a TMS (trimethylsilyl group).

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HMDS ( Hexamethyldisilzane ) TMCS ( Trimethylchlorosilane ) BSA ( Bistrimethylsilylacetamide ) Derivatization reaction of androsterone using TMSI/ methoxyamine .

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Alkylation: Alkylating reagents target active hydrogen's on amines and acidic hydroxyl groups. The principal reaction employed for preparation of these derivatives is nucleophilic displacement. DMF ( dialkylacetals ) TBH ( tetrabutylammonium hydroxide) PFBB ( Pentafluorobenzyl bromide) Acylation Acylating reagents react with highly polar functional groups such as amino acids or carbohydrates.

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Acylation reduces the polarity of amino, hydroxyl, and thiol groups and adds halogenated functionalities for ECD. Fluorinated Anhydrides Fluoracylimidazoles MBTFA-N-Methyl- bis ( trifluoroacetamide )

BASIC STEPS INVOLVED IN DEVELOPING SEPARATION METHODS::

BASIC STEPS INVOLVED IN DEVELOPING SEPARATION METHODS: Method development is the process of determining what conditions are adequate or ideal for the analysis. Obtained as much information as possible define separation objectives/goals Identify what separation technique is the most suitable Is sample preparation/sample pre-treatment required Choose detector mode and detector settings

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Optimize separation conditions Choose method, carry out preliminary run; estimate best separation conditions Check for problems or requirements for special procedures. Quantitative Qualitative Validate method for release to routine laboratory

Steps involved in gc method development:

Steps involved in gc method development Selection of stationary phase Selection of column Selection of carrier gas Selection of injection mode Selection of temperature Selection of methods Selection of flow rate

VALIDATION:

VALIDATION The principle purpose of analytical validation is to ensure that a selected analytical procedure will give reproducible and reliable results that are adequate for the intended purpose. It is thus necessary to define properly both the conditions in which the procedure is to be used and the purpose for which it is intended.

BASIC PARAMETERS FOR THE METHOD VALIDATION:

BASIC PARAMETERS FOR THE METHOD VALIDATION

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Accuracy: Definition: The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or as an accepted reference value and the value found. %relative substance (calculated) Overall accuracy = ----------------------------------------- %relative substance (theory)

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RANGE The range of analytical procedure is the interval between the upper and lower concentrations of analytes for which it has been demonstrated that the analytical procedure has suitable level of precision, accuracy, and linearity . LINEARITY: Definition : linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration of analytes in the sample. If there is a linear relationship test results should be evaluated by appropriate statistical methods like, correlation coefficient y-intercept

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Precision : Definition : The Precision is a measure of the ability of the method to generate reproducible results. The precision of a method is evaluated for repeatability, intermediate precision, and reproducibility. 21

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Limit of Detection (LOD, DL): The LOD of an analytical procedure is the lowest amount of analyte in sample which can be detected but not necessarily quantitated as an exact value. Limit of Quantitation (LOQ, QL) The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.The quantitation limit is used particularly for the determination of impurities and/or degradation products.

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DEVELOPMENT AND VALIDATION OF A GAS CHROMATOGRAPHIC METHOD FOR THE ASSAY OF MEMANTINE HYDROCHLORIDE IN PURE AND TABLET DOSAGE FORMS: Memantine (1-amino-3,5-dimethyladamantane) is a tricyclic amine structurally and pharmacologically related to the antiviral Amantadine. This drug is used to treat Parkinson's disease and movement disorders, as well as dementia syndrome.

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Preparation of stock and standard . The stock solutions of MMT and GPN ( gabapentine ) were prepared separately by dis - solving 2.5 g of MMT and 1.0 g of GPN in 50 mL of methanol. Preparation of tablet samples. Ten tablets of MMT were weighted and finely powdered. An accurately weighed quantity of the powdered tablet contents equivalent to 100 mg of the active ingredient was transferred into a 100 mL beaker.

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GC/FID ANALYSIS A GC 17A (SHIMANDZU) A fused silica capillary column DB-624 Mobile phase: Nitrogen gas Flow rate: 40 mL /min. column temperature: 300 c

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Linearity Linearity and range of the developed method were determined by analyzing different concentrations of the mixed standard solution containing 0.3-3.0 mg/ mL of MMT and 20 mg of GPN under the chromatographic conditions. The response factor of the standard solutions was calculated. The ratio of peak area of MMT to that of GPN was plotted against the concentration of MMT to obtain the calibration graph and was found to be linear over the concentration range of 0.5-3.5 mg/ mL of MMT.

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Parameters GPN MMT Retention time ( tR ) 5.0 8.7 Capacity factor (k1) 2.75 1.12 Peak asymmetry (T) 0.4 1.0 Selectivity factor () 0.567 Resolution (Rs) 52.0 Height equivalent to theoretical plate (HETP) in mm 4.14 ·10−6 System performance parameters for MMT and GPN Parameters MMT Linear dynamic range [mg/ mL ] 0.5-3.5 Regression equation (Y)* Slope (b 0.059 Intercept (a) 0.425 Correlation coefficient (R2) 0.9990 RSD [%] 1.24 Error [%] −0.78 LOD [mg/ mL ]) 0.79 LOQ [mg/ mL ]) 2.63 8 Y = bX + c , where 'Y' represents the ratio of MMT peak area to GPN peak area and 'X' represents the concentration of MMT. Linear regression least squares fit data for the determination of MMT

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CONCLUSION This study showed that the antiviral drug. MMT can be precisely and accurately determined in pure and pharmaceutical dosages . system performance parameters revealed that the method is ideal for the assay of MMT.

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Gas Chromatographic Method Development and Validation ofAssay Method for the Determination of Ticlopidine Hydrochloride in Tablets Formulation Ticlopidine hydrochloride is chemically 5-[(2-Chlorophenyl) methyl]-4, 5, 6, 7-tetrahydrothieno [3, 2-c] pyridine hydrochloride. Its molecular formula is C14H14ClNS.

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CHROMATOGRAPHIC CONDITIONS INJECTOR TEMPERATURE : 290˚C COLOUMN : SGE C5 BP ˣ 50 CARRIER GAS : Nitrogen CARRIER GAS PRESSURE : 200KPa COLOUMN OVEN TEMP : isothermal at 270 ˚c INJECTION VOLUME : 3µl DETECTOR : FID

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Development and Optimization of the GC Method Ticlopidine hydrochloride is soluble in polar solvent hence gas chromatography was selected to estimate them. To develop a rugged and suitable GC method for the quantitative determination of ticlopidine hydrochloride, the analytical condition were selected after testing the different parameters such as diluents, melting point and other chromatographic conditions.

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Method validation Linearity: Seven points calibration curve were obtained in a concentration range from 1000-4000 μg /ml for ticlopidine hydrochloride. A stock Solution of 12000 μg /ml was used for preparation of linearity range solution. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 412,673.29x - 61,103.79 with correlation coefficient 0.9980.

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LOD and LOQ The limit of detection and limit of quantification were evaluated by serial dilutions of ticlopidine hydrochloride stock solution in order to obtain signal to noise ratio of 3:1 for LOD and 10:1 for LOQ. The LOD value for ticlopidine hydrochloride was found to be 0.025 μg/ml and the LOQ value 0.25 μg/ml.

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Precision : The developed method was found to be precise as the %RSD values for the repeatability and intermediate precision studies were 0.83% and 0.42%, respectively, which conforms that method was precise. Accuracy: accuracy study was conducted by using standard addition method. A known concentration of standard substance was added to blank preparation of sample matrix and recovery of analyte is calculated on the basis of area obtained in the chromatogram The result shows that best recoveries (99.33-101.37 %) of the spiked drug were obtained at each added concentration , indicating that the method was accurate.

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Level % Amount added conc (mg/ml) Amount found conc %Recovery %RSD 50 1.25100 1.24257 99.33 0.38 100 2.50133 2.53567 101.37 0.42 150 3.75167 3.79454 101.14 0.38

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Conclusion This GC method for assay of Ticlopidine hydrochloride in a tablet formulation was successfully developed and validated for its intended purpose. The method was shown to specific, linear, precise, accurate, and robust. Because the method separates Ticlopidine hydrochloride and all the degradation products formed under variety of stress conditions it can be regarded as stability indicating.

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REFERENCES Instrumental method of chemical analysis Gurdeep , R.Chatwal , sham, K. Anand Gas liquid chromatography . Fifth edition-2002. page no 2.673-2.684. Instumental analysis Skoog , Holler , Nieman GC chromatography 5 th edition pg-no;702-715. RESTEK innovative chromatography solutions gas-liquid chromatography. Blau K. King, G . Hand book of Derivatives for Chromatography . Heyden & Sons Ltd.; 1979

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International journal of chemical studies gas chromatography kapil L.Dubal , Vijay R. Ram Govind J . Kher 2013 Vol. 1 No.3 page no 101-106. K.Siddappa , Metre Mallikarjun , Chandrakanth gas chromatography vol.9,2011,page.no.1-8.

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Thank you…….!!!

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