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MOST WIDELY USED METHOD FOR SEPARATION OF DIFFERENT TYPES OF DRUGS.A 20MICROLETERS SAMPLE IS SAFFICIANT TO SEPARATION OF DRUG.

MOST WIDELY USED METHOD FOR SEPARATION OF DIFFERENT TYPES OF DRUGS.A 20MICROLETERS SAMPLE IS SAFFICIANT TO SEPARATION OF DRUG.

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  • 1. Presented by G.Krishnam Raju M.Pharmacy Pharmaceutical Analysis & Quality Assurance 1st year 2nd semester HT.NO:-13TK6S0401 SVS Group Of Institutions SVS school of pharmacy
  • 2. CONTENT:-  INRODUCTION  HPLC METHOD DEVELOPMENT STEPS  CONCLUSION  REFERENCE
  • 3. 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 . 28 January 2015 3
  • 4. There are several reasons for developing new methods of analysis: 1. A suitable method for particular analyte in the specific matrix is not available. 2. Existing methods may be too error or they may be unreliable (have poor accuracy or precision) 3. Existing methods may be too expensive, time consuming. 28 January 2015 4
  • 5. 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. 28 January 2015 5
  • 6. Step 3a Initial HPLC condition Step 2 Sample preparationStep 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. 28 January 2015 6
  • 7. 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 ; a. sample preparation b. Types of chromatography c. Column selection d. Detector selection e. Selection of mobile phase composition 28 January 2015 7
  • 8. 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 : 1. It is relatively free of interferences 2. Will not damage the column 3. Should compatible with the intended HPLC method ; 28 January 2015 8
  • 9. 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. 28 January 2015 9
  • 10.  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. 28 January 2015 10
  • 11. COLUMNSELECTION 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. 28 January 2015 11
  • 12. Column selection 28 January 2015 12 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? • pKa values of sample components? • Concentration range? • Molecular weight range? • Solubility? • Other pertinent data? Column Chemistry (bonded phase, bonding type, endcapping, carbon load)
  • 13. Packing material:-  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. 28 January 2015 13
  • 14. • 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. 28 January 2015 14
  • 15. 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 28 January 2015 15
  • 16. 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. 28 January 2015 16
  • 17. 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. 1. Dynamic range 2. Response index or linearity 3. Linear dynamic range 4. Detector response 5. Detector noise level 6. Detector sensitivity, or minimum detectable concentration. 7. Total system dispersion 8. Pressure sensitivity 9. Flow arte sensitivity. 10. Operating temperature range. 28 January 2015 17
  • 18. 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: 28 January 2015 18 S.NO CHARACTERISTIC FIXED WAVELENGTH UV DETECTOR DIODE ARRAY DETECTOR 1 Sensitivity (solute benzene) 5×10ˉ⁸gml 1×10ˉ⁷gml 2 Linear dynamic range 5×10ˉ⁸ to 5×10ˉ⁴gml 10ˉ⁸to 5×10ˉ⁴gml 3 Response index 0.98 - 1.02 0.97- 1.03
  • 19. 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: 28 January 2015 19 S.No CHARACTERISTIC FLUOROSCENCE DETECTOR 1. Sensitivity (solute anthracene) 1×10ˉ⁹gml 2 Linear dynamic range 1×10ˉ⁹ to 5×10ˉ⁶gml 3 Response index 0.96 – 1.04
  • 20. 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: 28 January 2015 20 S.NO CHARACTERISTIC CONDUCTIVITY DETECTOR 1. Sensitivity(sodium chloride) 5×10ˉ⁹gml 2. Linear dynamic range 5×10ˉ⁹to 1×10ˉ6gml 3. Response index 0.97-1.03
  • 21. 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: 28 January 2015 21 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
  • 22. 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. 28 January 2015 22
  • 23.  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. 28 January 2015 23
  • 24. 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 28 January 2015 24
  • 25. 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. 28 January 2015 25
  • 26. 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. 28 January 2015 26
  • 27. 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. 28 January 2015 27
  • 28. 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. 28 January 2015 28
  • 29. By usINg soft wares :  Chemometrics in HPLC Optimization  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 2928 January 2015
  • 30. STEP 5 METHOD VALIDATION 28 January 2015 30
  • 31.  The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. 28 January 2015 31
  • 32.  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. 28 January 2015 32
  • 33. • 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. 28 January 2015 33
  • 34. 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 28 January 2015 34
  • 35. 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. 28 January 2015 35
  • 36. 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 28 January 2015 36
  • 37. 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. 28 January 2015 37
  • 38. 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. 28 January 2015 38
  • 39. 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 28 January 2015 39
  • 40. 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. 28 January 2015 40
  • 41. 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 28 January 2015 41
  • 42. 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. 28 January 2015 42
  • 43. 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. 28 January 2015 43
  • 44. 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 28 January 2015 44
  • 45. 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: 28 January 2015 45
  • 46. 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; 28 January 2015 46
  • 47. 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. 28 January 2015 47
  • 48. CONCLUSION  The method development and validation are continuous and interrelated processes that are conducted throughout the drug development process.  The analytical validation verifies that a given method measures a parameter as intended and establishes the performance limits of the measurement.  Reproducible quality HPLC results can only be obtained if proper attention has been paid to the method development, validation and system’s suitability to carry out the analysis.
  • 49. references  Instrumental methods of chemical analysis by Gurdeep.R.Chatwal, Sham.K Anand, p.no 2.624-2.639.  D. H. Shewiy, E. Kaale, P. G. Risha, B. Dejaegher, J. S. Verbeke, Y. V. Heyden, J. Pharmaceut. Biomed. Anal 2012, 66, 11–23.  M. D. Rockville, General Tests, Chapter 621 – Chromatography System Suitability, United States Pharmacopeial Convention (USP), USP 31 (2009):  Kasawar GB, Farooqui M. Development and validation of a stability indicating RP-HPLC method for the simultaneous determination of related substances of albuterol sulfate and ipratropium bromide in nasal solution. J Pharmaceut Biomed Anal 2010; 52:19–29 28 January 2015 49
  • 50. Thank you 28 January 2015 50