HPLC method development,optimizationand validation

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hplc is a widely used techniqui in analysis of samples

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METHOD DEVELOPMENT,OPTIMIZATION AND VALIDATION OF HPLC:

METHOD DEVELOPMENT,OPTIMIZATION AND VALIDATION OF HPLC PRESENTED BY: UNDER THE GUIDANCE OF: Cheedirala.Swathi Dr.V.Sree janardanan I year M.pharmacy M.pharm., P.hD 2 semister (pharmaceutical analysis) 1 10 September 2013

CONTENTS:

CONTENTS Introduction. Modes of separation. Instrumentation. HPLC method development,optimization and validation. References. 2 10 September 2013

INTRODUCTION:

INTRODUCTION Most of the drugs in multicomponent dosage forms can be analyzed by HPLC method because of the several advantages like: Improved resolution of the separated substances Faster separation times and The improved accuracy, precision, & sensitivity with which the separated substances may be quantified. 3 10 September 2013

MODES OF SEPARATION IN HPLC:

MODES OF SEPARATION IN HPLC There are different modes of separation in HPLC: Normal phase mode. Reversed phase mode. Reversed phase ion pair chromatography. Affinity chromatography. Size exclusion chromatography 4 10 September 2013

Normal phase mode :

Normal phase mode Stationary phase-polar in nature. Mobile phase-non polar in nature. Generally used for separation of non polar compounds. 5 10 September 2013

Reversed phase mode:

Reversed phase mode Stationary phase-non polar . Mobile phase-polar in nature. Generally used for separation of polar compounds . 6 10 September 2013

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ION EXCHANGE CHROMATOGRAPHY : The stationary phase contains ionic groups like NR⁺ з , SO⁻ з which interact with the ionic groups of the sample molecules. This method is suitable for the separation of charged molecules only. ION PAIR CHROMATOGRAPHY : This may be used for the separation of ionic compounds. Strong acids & basic compounds may be separated by reversed phase mode by forming ion pairs with suitable counter ions. 7 10 September 2013

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AFFINITY CHROMATOGRAPHY: It uses highly specific biochemical interactions for separations. The stationary phase contains specific groups of molecules which can absorb the sample if certain steric & charge related conditions are satisfied. This technique can be used to isolate proteins, enzymes, as wel as antibodies from complex mixture. SIZE EXCLUSION CHROMATOGRAPHY : Separates molecules according to their molecular mass. Largest molecules are eluted first and smaller molecules last. 8 10 September 2013

PRINCIPLE OF SEPARATION:

PRINCIPLE OF SEPARATION The principle of separation is adsorption. Separation of components takes place because of the difference in affinity of compounds towards stationary phase. 9 10 September 2013

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INSTRUMENTATION 10 10 September 2013

VARIOUS COMPONENTS OF HPLC:

VARIOUS COMPONENTS OF HPLC A solvent delivery system including pump. Sample injection system A chromatographic column Detectors Recorders and integrators 11 10 September 2013

A SOLVENT DELIVERY SYSTEM:

A SOLVENT DELIVERY SYSTEM A mobile phase is pumped under pressure from one or several reservoir and flows through the column at a constant rate. For normal phase separation eluting power increases with increasing polarity of the solvent but for reversed phase separation, eluting power decreases with increasing polarity. A degasser is needed to remove dissolved air and other gases from the solvent. 12 10 September 2013

Varian solvent delivery systems:

Varian solvent delivery systems 13 10 September 2013

PUMPS:

PUMPS The pump is one of the most important component of HPLC, since its performance directly affects retention time, reproducibility and detector sensitivity. Three main types of pumps are used in HPLC. Displacement pump Reciprocating pump Pneumatic or constant pressure pump 14 10 September 2013

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DISPLACEMENT PUMP : It produce a flow that tends to independent of viscosity and back pressure and also output is pulse free but possesses limited capacity (250ml). RECIPROCATING PUMP : It has small internal volume (35-400µl), their high output pressure(up to 10,000psi) and their constant flow rates. But it produces a pulsed flow. PNEUMATIC OR CONSTANT PRESSURE PUMP : They are pulse free . Suffer from limited capacity as well as a dependence of flow rate on solvent viscosity and column back pressure. They are limited to pressure less than 2000psi. 15 10 September 2013

SAMPLE INJECTION SYSTEM:

SAMPLE INJECTION SYSTEM There are three important ways of introducing the sample in to the injection port. Loop injection : in which a fixed amount of volume is introduced by making use of fixed volume loop injector. Valve injection : in which, a variable volume is introduced by making use of an injection valve. On column injection : in which, a variable volume is introduced by means of a syringe through a septum. 16 10 September 2013

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Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H 2 O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject 18 10 September 2013

CHROMATOGRAPHIC COLUMN:

CHROMATOGRAPHIC COLUMN The column is usually made up of heavy glass or stainless steel tubule to withstand high pressure . The columns are usually 10-30cm long and 4-10mm inside diameter containing stationary phase at particle diameter of 25µm or less. Column with internal diameter of 5mm give good results because of compromise between efficiency, sample capacity, and the amount of packaging and solvent required. 19 10 September 2013

DETECTORS:

DETECTORS The function of detector in HPLC is to monitor the mobile phase as it merges from the column. Detectors are usually of two types: 1. Bulk property detectors : It compares overall changes in a physical property of the mobile phase with and without an eluting solute e.g. refractive index ,dielectric constant or density. 2.Solute property detectors : It responds to a physical property of the solute which is not exbited by the pure mobile phase.e.g.UV absorbance,fluoroscence or diffusion current. 20 10 September 2013

TYPES OF DETECTORS:

TYPES OF DETECTORS There are mainly 4 types of detectors are used in HPLC: 1. Photometric detectors. Single wavelength detectors. Multiwavelength detectors. Variable wavelength detectors. Programmable detectors. Diode array detectors . 2. Fluorescence detectors. 3. Refractive index detectors. 4. Electrochemical detectors. 21 10 September 2013

PHOTOMETRIC DETECTORS:

PHOTOMETRIC DETECTORS These normally operate in the ultra violet region of the spectrum . Most extensively used in pharmaceutical analysis. 22 10 September 2013

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SINGLE WAVELENGTH DETECTORS Equipped with a low pressure mercury discharge lamp. The absorbance is measured at the wavelength of mercury at 254nm. 23 10 September 2013

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MULTOWAVELENGTH DETECTORS Employ mercury and other discharge sources. When used in combination with interference filters ,allow a no of monochromatic wavelengths to be selected e.g. 206, 226, 280 , 313, 340 or 365 nm. 24 10 September 2013

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Multi-wavelength UV-Vis Absorption Detector Deuterium Lamp Photodiode Array 25 10 September 2013

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VARIABLE WAVELENGTH DETECTORS Use a deuterium light source. A grating monochromator to allow selection of any wavelength in deuterium continuum (190-360nm). 26 10 September 2013

Variable wavelength detector:

Variable wavelength detector 27 10 September 2013

Variable UV/Vis Detector:

Variable UV/Vis Detector ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready 28 10 September 2013

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PROGRAMMABLE DETECTORS Allow the automatic change of wavelength between and during the chromatographic analysis. 29 10 September 2013

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DIODE ARRAY DETECTORS They are microprocessor – controlled photodiode array spectrophotometers in which light from an UV source passes through the flow cell into a polychromator which disperses the beam so that the full spectrum falls on the array of diodes. 30 10 September 2013

DIODE ARRAY DETECTOR:

DIODE ARRAY DETECTOR 31 10 September 2013

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FLUORESCENCE DETECTORS These are essentially filter fluorimeter or spectro -fluorimeters equipped with grating monochromators, and micro flow cell. Their sensitivity depends on the fluorescence properties of the components in the eluate. 32 10 September 2013

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Fluorescence Detector 33 10 September 2013

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REFRACTIVE INDEX DETECTORS Which respond to the change in the bulk property of the refractive index of the solution of the component in the mobile solvent system. The sensitivity of the refractive index detector is much less than that of specific solute property detectors, they are useful for the detection of substances( e.g ,carbohydrates& alcohols) which do not exhibit other properties that can be used as the basis for specific detection. 34 10 September 2013

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Refractive Index Detector 35 10 September 2013

ELECTROCHEMICAL DETECTORS:

ELECTROCHEMICAL DETECTORS These are based on standard electrochemical principles involving amperometry,voltametryand polarography . These detectors are very sensitive for substances that are electroactive ,i.e. those that undergo oxidation or reduction . They have found particular application in the assay of low levels of endogenous catecholamines in biological tissues,pesticides,tryptophan derivatives and many drugs. 36 10 September 2013

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Electrochemical Detector 37 10 September 2013

hplc METHOD DEVELOPMENT, optimization AND VALIDATION for pharmaceuticals:

hplc METHOD DEVELOPMENT, optimization AND VALIDATION for pharmaceuticals 38 10 September 2013

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Introduction: ANALYTICAL METHOD DEVELOPMENT: Method development usually requires selecting the method requirements and deciding on what type of instrumentation to utilize and why. The wide variety of equipment, columns, eluent and operational parameters involved makes HPLC method development . 39 10 September 2013

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There are several reasons for developing new methods of analysis: A suitable method for particular analyte in the specific matrix is not available. Existing methods may be too error or they may be unreliable (have poor accuracy or precision) Existing methods may be too expensive, time consuming. 40 10 September 2013

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HPLC method development generally follows the following steps: Step 1- selection of the HPLC method and initial system. Step2- Selection of optimum conditions. Step3- selectivity optimization. Step4- system parameter optimization. Step5- method validation. 41 10 September 2013

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Step 3a Initial HPLC condition Step 2 Sample preparation Step 1 Method goals and chemistry Step 3b Optimize HPLC separation Step 4 Standardization Step 5 Method validation Fig 2.2 Pie diagram showing the time that should be spent on different steps of the method development to meet the commended timeline. The sequence of events and percentage of time allocated is only suggestive. 42 10 September 2013

STEP1-SELECTION OF HPLC METHOD AND INITIAL CONDITIONS:

STEP1-SELECTION OF HPLC METHOD AND INITIAL CONDITIONS SELECTION OF HPLC METHOD: When selecting an HPLC system it must have a high propability of actually being able to analyse the sample. For example if the sample includes polar analytes then RP-HPLC would offer both adequate retention and ressolution. consideration must be given to the following ; sample preparation Types of chromatography Column selection Detector selection Selection of mobile phase composition 43 10 September 2013

SAMPLE PREPARATION:

SAMPLE PREPARATION Sample preparation is an essential part of HPLC analysis, to provide a reproducible and homogenous solution i.e. suitable for injection on to the column. The aim of sample preparation is a sample that : It is relatively free of interferences Will not damage the column Should compatible with the intended HPLC method ; 44 10 September 2013

TYPES OF CHROMATOGRAPHY:

TYPES OF CHROMATOGRAPHY Reversed phase is the choice for the majority of the samples. But if acidic or basic analytes are present reversed phase ion suppression (for weak acids and bases) or reversed phase ion pairing (for strong acids and bases) should be used. For low or medium polarity analytes normal phase HPLC is used, particularly if the separation of isomers is required. For inorganic anion or cation analysis ion exchange chromatography is best. 45 10 September 2013

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Size exclusion chromatography would normally be considered for analyzing high molecular weight compounds. Gradient HPLC only a requirement for complex samples with a large number of components (20-30) . Reversed phase HPLC is commonly used in peptide and small protein analysis using an acetonitrile –water mobile phase containing 1% trifluoroethanolic acid. 46 10 September 2013

COLUMN SELECTION:

COLUMN SELECTION A column is chosen based on the Knowledge of sample On the expectation of how its components will interact with the packing material. the properties of column packing material. 47 10 September 2013

Column selection :

Column selection 1.Knowledge of the Sample : which influences the choice of Column Bonded Phase characteristics Knowledge of the sample • Structure of sample components? • Number of compounds present? • Sample matrix? • pK a values of sample components? • Concentration range? • Molecular weight range? • Solubility? • Other pertinent data? Column Chemistry (bonded phase, bonding type, endcapping, carbon load) } 48 10 September 2013

Pac king material:-:

Pac king material:- 10 September 2013 49 Most HPLC separations are performed on bonded phase HPLC columns Octadecyl-derivatized silica gel columns are the most widely used bonded phase columns in the reverse phase mode. Commonly used polar bonded phases certain diol,cyano or amino functional groups. Silica based packing materials are used in about 75% of all HPLC separations performed today.

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due to the physical stability the availability of bonded phase and to the high efficiency of silica based HPLC columns Many new resin packings have been introduced in recent years particularly for biochemical analysis. The most well known resin based packings are formed by the co polymerization of polysterene and divinyl benzene. Other packing materials such as alumina, titania and zirconia have also been employed for bio polymer analysis. 50 10 September 2013

COLUMN DIMENSIONS:

COLUMN DIMENSIONS Effect on chromatography Column Dimension • Short (30-50mm) - short run times, low backpressure • Long (250-300mm) - higher resolution, long run times • Narrow (  2.1mm) - higher detector sensitivity • Wide (10-22mm) - high sample loading 51 10 September 2013

DETECTOR SELECTION:

DETECTOR SELECTION Consideration must given to the following: Do the analytes have chromophores to enable UV detection ? Is more selective or sensitive detection required? What detection limits are necessary ? Will the sample require chemical derivatization to enhance detect ability and /or improve the chromatography. 52 10 September 2013

DETECTOR SELECTION :

DETECTOR SELECTION A HPLC detector will have a number of performance characteristics that need to be specified and known before a particular detector can be chosen for a specific application and are listed as follows. Dynamic range Response index or linearity Linear dynamic range Detector response Detector noise level Detector sensitivity, or minimum detectable concentration. Total system dispersion Pressure sensitivity Flow arte sensitivity. Operating temperature range. 53 10 September 2013

THE UV DETECTOR:

THE UV DETECTOR Limited to the detection of those substances that absorb light in the UV wave length range. UV detectors detects all sample components that contain chromophores. Specifications: S.NO CHARACTERISTIC FIXED WAVELENGTH UV DETECTOR DIODE ARRAY DETECTOR 1 Sensitivity (solute benzene) 5×10ˉ⁸g\ml 1×10ˉ⁷g\ml 2 Linear dynamic range 5×10ˉ⁸ to 5×10ˉ⁴g\ml 10ˉ⁸to 5×10ˉ⁴g\ml 3 Response index 0.98 - 1.02 0.97- 1.03 54 10 September 2013

THE FLUORESCENCE DETECTOR:

THE FLUORESCENCE DETECTOR It can detect eluted solutes on the basis of fluorescence ,but it can also provide their fluorescence spectra. Fluorescence and electro chemical detectors should be used for trace analysis. Specifications: S.No CHARACTERISTIC FLUOROSCENCE DETECTOR 1. Sensitivity (solute anthracene) 1×10ˉ⁹g\ml 2 Linear dynamic range 1×10ˉ⁹ to 5×10ˉ⁶g\ml 3 Response index 0.96 – 1.04 55 10 September 2013

ELECTRICAL CONDUCTIVITY DETECTOR:

ELECTRICAL CONDUCTIVITY DETECTOR It is usually employed with an ion suppressor column to allow salts and buffers to be used in the mobile phase without affecting the detector output. Specifications: S.NO CHARACTERISTIC CONDUCTIVITY DETECTOR 1. Sensitivity(sodium chloride) 5×10ˉ⁹g\ml 2. Linear dynamic range 5×10ˉ⁹to 1×10ˉ6g\ml 3. Response index 0.97-1.03 56 10 September 2013

REFRACTIVE INDEX DETECTOR:

REFRACTIVE INDEX DETECTOR It is one of the least sensitive LC detectors and is used circumstances where other detector s are inappropriate. For preparative HPLC it is preferred because it can handle concentration without overloading the detector Specifications: S.NO CHARACTERISTIC RI DETECTOR 1. Sensitivity(solute benzene ) 1×10ˉ⁶g/ml 2. Linear dynamic range 1×10ˉ⁶ -1×10ˉ⁴g/ml 3. Response index 0.97-1.03 57 10 September 2013

MOBILE PHASE SELECTION:

MOBILE PHASE SELECTION The organic phase concentration required for the mobile phase can be estimated by gradient elution method. Gradient can be started with 5-10 % of the organic phase in the mobile phase and the organic phase concentration can be increased up to 100% within 30-45%. The elution strength of a mobile phase depends upon its polarity, the stronger the polarity higher is the elution. 58 10 September 2013

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Ionic samples(acidic or basic) can be separated if they are present in undissociated form. Dissociation of ionic samples may be suppressed by the selection oh pH. If the retention times are too long an increase of the organic phase concentration is needed. When tailing or fronting is observed, it means that the mobile phase is not totally compatible with the solutes . 59 10 September 2013

SELECTION OF INITIAL SYSTEM:

SELECTION OF INITIAL SYSTEM It could based on : assessment of the nature of the sample and analytes together with literature data. Experience Expert system software and Empirical approaches 60 10 September 2013

STEP 2 : SELECTION OF INITIAL CONDITIONS:

STEP 2 : SELECTION OF INITIAL CONDITIONS This step determines the optimum conditions to adequately retain all analytes ; i.e. Ensures no analyte has a capacity factor of less than 0.5(poor retention could result in peak overlapping). No analyte has a capacity factor greater than 10-15 (excessive retention leads to long analysis time and broad peaks with poor detectability). Determination of initial conditions: The recommended method involves performing two gradient runs differ in only in the run time. A binary system based on either aceto nitrile/ water or methanol/water should be used. 61 10 September 2013

STEP 3: SELECTIVITY OPTIMIZATION:

STEP 3: SELECTIVITY OPTIMIZATION The aim of this step is to achieve adequate selectivity. The mobile phase and stationary phase compositions need to be taken in to account. To select these the nature of the analytes must be considered. Once the analyte types are identified the relevant optimization parameters may be selected. 62 10 September 2013

STEP 4: SYSTEM PARAMETER OPTIMIZATION:

STEP 4: SYSTEM PARAMETER OPTIMIZATION This is used to find the desired balance between ressolution and analysis time after satisfactory selectivity has been achieved. The parameters involve include column dimensions, column packing particle size and flow rate. This parameters may be changed without affecting capacity factors or selectivity. 63 10 September 2013

TYPES OF OPTIMIZATION:

TYPES OF OPTIMIZATION Two types : Manually and By using soft wares. By Manual : separation then can be optimized by change in the initial mobile phase composition and the slope of the gradient according to the chromatogram obtained from the preliminary run. 64 10 September 2013

Chemometrics in HPLC Optimization :

Chemometrics in HPLC Optimization By usINg soft wares : 10 September 2013 65 Chemo metric protocols available for the development and optimization of HPLC methods : Experimental Design (ED) Factorial design Plackett-Burman design D-optimal design Two-level full factorial design Central composite design Box- Behnken design Doehlert design Multi-Criteria Decision Making (MCDM) Overlay plots Pareto optimality Utility function Derringer’s desirability function

STEP 5 METHOD VALIDATION:

STEP 5 METHOD VALIDATION VALIDATION PARAMETERS BASED ON ICH GUIDELINES 66 10 September 2013

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The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. 67 10 September 2013

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A brief description of types of tests considered in this document is provided below:- Identification tests are intended to ensure the identity of analyte in a sample. This normally achieved by comparing the properties of sample with that of the reference standard. Testing for impurities can be either a quantitative test or a limit test for the impurity in a sample. Assay procedures are intended to measure the analyte present in a given sample. 68 10 September 2013

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Typical validation characteristics which should be considered are listed below:- 1.Accuracy 2. Precision a. repeatability b. intermediate precision 3. specificity 4. Detection limit 5. Quantitation limit 6. Linearity 7. range. 69 10 September 2013

ACCURACY:

ACCURACY The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true or an accepted reference value and the value found. Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range 70 10 September 2013

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LINEARITY The linearity of an analytical procedure is its ability to obtain test results which are directly proportional to the concentration of analyte in the sample. Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. For the establishment of linearity, a minimum of 5 concentrations is recommended. 71 10 September 2013

PRECISION:

PRECISION The precision of an analytical procedure expresses the closeness of agreement between the measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. repeatability Precision intermediate precision reproducibility 72 10 September 2013

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Repeatability :- Expresses the precision under the same operating conditions over a short interval of time . Repeatability is also termed intra- assay precision. a) a minimum of 9 determinations covering the specified range for the procedure (e.g., 3 concentrations/3 replicates each); or b) a minimum of 6 determinations at 100% of the test concentration. 73 10 September 2013

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Intermediate precision :- intermediate precision expresses within – laboratories variations: different days, different analysts, different equipment e.t.c. Reproducibility:- reproducibility expresses the precision between laboratories. The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated. 74 10 September 2013

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DETECTION LIMIT The detection limit of individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable 75 10 September 2013

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Based on Visual Evaluation Visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods. The detection limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. Based on Signal-to-Noise This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. 76 10 September 2013

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Based on the Standard Deviation of the Response and the Slope The detection limit (DL) may be expressed as: DL = 3.3 σ /s Where, σ = the standard deviation of the response S = the slope of the calibration curve 77 10 September 2013

QUANTITATION LIMIT:

QUANTITATION LIMIT The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and or degradation products. 78 10 September 2013

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Based on Visual Evaluation Visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods. The detection limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. Based on Signal-to-Noise This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte. A signal-to-noise ratio between 10:1 is generally considered acceptable for estimating the detection limit. 79 10 September 2013

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Based on the Standard Deviation of the Response and the Slope The quantitation limit (QL ) may be expressed as: QL = 10 σ /s Where, σ = the standard deviation of the response S = the slope of the calibration curve 80 10 September 2013

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RANGE The range of an analytical procedure is the interval between the upper and lower concentration of the analyte in the sample for which it has been demon started that the analytical procedure has a suitable level of precision, accuracy and linearity. The following minimum specified ranges should be considered: 81 10 September 2013

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for the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration; for content uniformity, covering a minimum of 70 to 130 percent of the test concentration, unless a wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified; for dissolution testing: +/-20 % over the specified range; 82 10 September 2013

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ROBUSTNESS The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberated variations in method parameters and provides an indication of its reliability during normal usage. 83 10 September 2013

references:

references High performance liquid chromatography; principles and methods in biotechnology by Elena.d katz . Practical pharmaceutical chemistry part 2 fourth edition by A.H.Beckett, J.B.stenlake; p.no 157. Instrumental methods of chemical analysis by Gurdeep.R.Chatwal, Sham.K Anand , p.no 2.624-2.639 . 84 10 September 2013

Thank you:

Thank you 85 10 September 2013

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