SEMINAR ON IMPURITY PROFILING OF API’s USING RP-HPLCFor the partial fulfillment of…MASTER DEGREE IN PHARMACYBYJ.N.V.INDIRA...
ContentsIntroductionObjectiveDescription:ICH Impurities guidelinesClassification of impuritiesICH limits for impurities...
Impurity as per International Conference on Harmonization…According to the ICH of TechnicalRequirements for the Registrat...
What is an impurity profile?‘A description of the identified and unidentifiedimpurities present in a new drug substance.1...
 Various regulatory authorities and Health Agency areemphasizing on the purity requirements and theidentification of impu...
ICH impurities guidelinesCodes TitleQ 3 A (R2)Dated 25th Oct 2006Impurities in new drugsubstancesQ 3 B (R2)Dated 2nd June ...
Classification of impurities2=>Organic(process and drug related)-includes starting materials, by-products, intermediates, ...
1. Organic ImpurityDegradation product The degradation of penicillins and cephalosporins is a well-known exampleof degrad...
3. Residual Solvents: Residual solvents are defined as the organic volatilechemicals that are used or produced in the man...
Class 1 Solvent: Solvent to be avoided• Solvents in class 1 should not be employed in the manufacturing ofdrug substance b...
Class 3 Solvent : Solvent with low toxic potential-Regarded as less toxic and of lower risk to human health.-No long term ...
Maximumdaily dose1ReportingThreshold2,3IdentificationThreshold3Qualificationthreshold3≤2g/day 0.05% 0.10% or 1.0mgper day ...
Impurity profiling by rp-hplcChromatographic impurity profiles are most often developed using (RP-HPLC).The chromatograp...
1.Selection of a Set of DissimilarChromatographic Columns To reveal and/or separate all impurities in a new drugsubstance...
HPLC Columns:Orthogonal Screening – Columns10StationaryPhaseaColumn pH Rangeb Manufacturer Part NumberC18 – TwinTechnology...
2.Optimization of the Mobile Phase pH and Selection of a SuitableColumn Fig.2 consists of first modelling the retention o...
5/4/2013 17
These Rsmin are plotted as a function ofthe pH values for each column (Figure 3).The pH value with maximal Rsmin, the on...
3.Optimization of the Organic Modifier CompositionModifier Mobile PhaseConcentrationApproximate pHTrifluoroacetic Acid (TF...
 In general, for a three-component mixture, for eg. with modifiers methanol(MeOH), acetonitrile (ACN) and tetrahydrofuran...
Fig.1 Solvents triangle, with x1, for instance,representing an ACN/H2O mixture,x2 MeOH/H2O, and x3 THF/H2O. Watercontent i...
 Here only one side of the triangle is explored (e.g., side 2 to 3 inFigure 1). Two different retention models can be con...
 In Figure 2, this is represented for four substances. For each intermediatemixture of organic modifiers, the retention i...
Figure 2: Optimization of a binary mixture of organic modifiers using (a) a linear model, and(b) a quadratic polynomial mo...
4.Optimization of the Gradient Slope and the Temperature This step concerns factors with less influence on theselectivity...
Impurity profiling of Famotidine in bulk drugs and pharmaceuticalformulations by RP-HPLC method using ion pairing agent11...
Fig.The chromatogram of Famotidine and its related impurities5/4/2013 27Dept.of pharmaceutical analysis
Numerous applications have been sought in the areas of drug designingand in monitoring quality, stability and safety of ph...
Isolation and characterization of impurities isrequired for acquiring and evaluating data that establishesbiological safet...
1. International Conference on Harmonisation of Technical Requirements for the Registration ofPharmaceuticals for Human Us...
5/4/2013 31Dept.of pharmaceutical analysis
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Indu...impurity profiling of api’s using rp hplc as per

  1. 1. SEMINAR ON IMPURITY PROFILING OF API’s USING RP-HPLCFor the partial fulfillment of…MASTER DEGREE IN PHARMACYBYJ.N.V.INDIRA DEVIREG NO:611235004011YALAMARTY PHARMACY COLLEGEUNDER GUIDANCE OFSri.J.V.L.N SESHAGIRI RAO, PROFESSORMr.N.K SAHOO, ASSISTANT PROFESSORANDHRA UNIVERSITY5/4/2013 1Dept.of pharmaceutical analysis
  2. 2. ContentsIntroductionObjectiveDescription:ICH Impurities guidelinesClassification of impuritiesICH limits for impuritiesImpurity profiling by RP-HPLCApplicationsConclusionReference5/4/2013 2Dept.of pharmaceutical analysis
  3. 3. Impurity as per International Conference on Harmonization…According to the ICH of TechnicalRequirements for the Registration of Pharmaceuticals for Human Use(ICH) guideline Q3A(R2) on impurities in new drug substances,1 animpurity is defined as any component of the new drug substance thatis not the chemical entity defined as the new drug substance.Impurities can be formed during drug synthesis, manufacturing,and/or storage.1,2Introduction5/4/2013 3Dept.of pharmaceutical analysis
  4. 4. What is an impurity profile?‘A description of the identified and unidentifiedimpurities present in a new drug substance.1Identified Impurity: An impurity for which a structural characterizationhas been achieved.Unidentified Impurity: An impurity for which a structural characterizationhas not been achieved and that is defined solely by qualitative analyticalproperties .2A general acceptance criteria of not more than 0.1 percent for any unspecifiedimpurity should be included.35/4/2013 4Dept.of pharmaceutical analysis
  5. 5.  Various regulatory authorities and Health Agency areemphasizing on the purity requirements and theidentification of impurities in API. Qualification of the impurities is the process of acquiringand evaluating data that establishes biological safety of anindividual impurity; thus, revealing the need and scope ofimpurity profiling of drugs in pharmaceutical research2.5/4/2013 5Dept.of pharmaceutical analysis
  6. 6. ICH impurities guidelinesCodes TitleQ 3 A (R2)Dated 25th Oct 2006Impurities in new drugsubstancesQ 3 B (R2)Dated 2nd June 2006Impurities in new drugproductQ 3 C (R3)Dated 17th July1997,revised Nov 2005Impurities: Guidelines forresidual solvents5/4/2013 6Dept.of pharmaceutical analysis
  7. 7. Classification of impurities2=>Organic(process and drug related)-includes starting materials, by-products, intermediates, degradationproducts, reagents, ligands and catalysts=>Inorganic-includes reagents, ligands and catalysts, heavy metals or other residualmetals, inorganic salts, other materials (eg. filter aids, charcoal etc.)=>Residual solvents-organic or inorganic Specifically doesn’t cover:1) extraneous contaminants more appropriately addressed under GMP;2) polymorphic forms3)Enantiomeric impurities These are addressed in other ICH guidelines eg. Q6A5/4/2013 7Dept.of pharmaceutical analysis
  8. 8. 1. Organic ImpurityDegradation product The degradation of penicillins and cephalosporins is a well-known exampleof degradation products. The presence of a ß-lactam ring as well as that of an amino group in theC6/C7 side chain plays a critical role in their degradation.2.Inorganic impurityReagent , ligands, and catalysts :• The chances of having this impurities are rare.Heavy metal:• The main sources of heavy metals are water used in process and thereactor, where acidification or acid hydrolysis takes place.• These impurities can easily avoided using demineralised water.5/4/2013 8Dept.of pharmaceutical analysis
  9. 9. 3. Residual Solvents: Residual solvents are defined as the organic volatilechemicals that are used or produced in the manufacture of drugsubstances or excipients or in the preparation of the drugproducts.Classification of Residual solvent:Solvents are evaluated for their possible risk to humanhealth and placed in to one of three classes as follows: Class 1 solvents : solvents to be avoided. Class 2 solvents : solvents to be limited. Class 3 solvents : solvents with low toxic potential. Other solvents (“class 4”) : no adequate toxicologicaldata.5/4/2013 9Dept.of pharmaceutical analysis
  10. 10. Class 1 Solvent: Solvent to be avoided• Solvents in class 1 should not be employed in the manufacturing ofdrug substance because of their unacceptable toxicity or their deleteriousenvironmental effect.Solvent Concentration in ppm concernBenzene 2 CarcinogenCarbontetrachloride 4 Toxic and environmentalhazard1,2 Dichloroethan 5 ToxicClass 2 Solvent: Solvent To Be Limited• Solvent in the following table should be limited in pharmaceuticalproduct because of their inherent toxicity.Solvent Permitted dailyexposure (mg/day)Concentration limit(ppm)Acetonitrile 4.1 4100Chlorobenzene 3.6 36005/4/2013 10Dept.of pharmaceutical analysis
  11. 11. Class 3 Solvent : Solvent with low toxic potential-Regarded as less toxic and of lower risk to human health.-No long term toxicity or carcinogenicity studies.-Less toxic in acute or short term studies and negative in Genotoxicitystudies.Eg .- acetic acid- acetone- anisole- 2-propanol- methyl acetate- ethyl ether.Other solvents (“class 4”): no adequate toxicological dataThe following solvent may also be of interest to manufacture of Drugsubstances or drug product.Eg.: - Isooctane- petroleum ether- methyl isopropyl ketone- trichloroacetic acid.5/4/2013 11Dept.of pharmaceutical analysis
  12. 12. Maximumdaily dose1ReportingThreshold2,3IdentificationThreshold3Qualificationthreshold3≤2g/day 0.05% 0.10% or 1.0mgper day intake(whichever islower)0.15% or 1.0mgper day intake(whichever islower)>2g/day 0.03% 0.05% 0.05%ICH Q3A(R2): LIMITS FOR IMPURITIES1[1] The amount of drug substance administered per day[2] Higher reporting thresholds should be scientifically justified[3] Lower thresholds can be appropriate if the impurity is unusually toxic5/4/2013 12Dept.of pharmaceutical analysis
  13. 13. Impurity profiling by rp-hplcChromatographic impurity profiles are most often developed using (RP-HPLC).The chromatographic impurity profile should allow detecting and separating all (un)identifiedimpurities in each new active compound. 4.Selection of dissimilarchromatographic columnsOptimisation of mobile phasepH and column selectionOptimisation of organicmodifier compositionOptimisation of gradientslope and temperatureDifferent steps when developing chromatographic drug impurity profiles5/4/2013 13Dept.of pharmaceutical analysis
  14. 14. 1.Selection of a Set of DissimilarChromatographic Columns To reveal and/or separate all impurities in a new drugsubstance, several chromatographic profiles with different selectivitieswill be created. In impurity profiling, the stationary phase largely influences theselectivity of the chromatographic system.5 Therefore, one possibility to obtain a set of profiles with differentselectivities is to use a set of dissimilar (or orthogonal) RP-HPLCcolumns to screen the drug impurities mixture. This selection can be based on several approaches,5-8 . It often ispreferred to use only silica-based columns, which are widely available.5/4/2013 14Dept.of pharmaceutical analysis
  15. 15. HPLC Columns:Orthogonal Screening – Columns10StationaryPhaseaColumn pH Rangeb Manufacturer Part NumberC18 – TwinTechnologyGemini C18, 5 μm,110A, 4.6 x 150 mm1-12 Phenomenex 00F-4435-E0Phenyl with Hexyl(C6) linker,endcappedLuna Phenyl-Hexyl, 3μm, 4.6 x 150 mm1.5-10 Phenomenex 00F-4256-E0C18-20% C loading Discovery HS-C18,3μm, 4.6 x 150 mm2-8 Supelco 569252-UC18 – polarembedded, hybridparticle with ShieldTechnologyXTerra RP18, 3.5 μm,4.6 x 150 mm1-12 Waters 186000442C18– silica Sunfire C18, 3.5 μm,4.6 x 150 mm2.8 Waters 186002554Pentafluorophenyl Curosil PFP, 3 μm,4.6 x 150 mm2-7.5 Phenomenex 00F-4122-E0a Other columns could be selected based on the compound properties.b Columns were screened only against mobiles phases within their compatible pH range.5/4/2013 15
  16. 16. 2.Optimization of the Mobile Phase pH and Selection of a SuitableColumn Fig.2 consists of first modelling the retention of the peaks as a function ofthe pH and also the peak width in case of isocratic elution. For gradient elution, the peak width can be considered constant and does notneed to be modelled. For a compound with acidic or basic properties, retention changes with pH,following a sigmoidal curve. However, depending on the pKa of theimpurities and the examined pH range, the whole curve is not considered.This makes modelling tR as a function of pH challenging.9 Resolutions between consecutive peaks are then calculated and the minimalresolution, Rsmin, determined at each pH value on the given column.5/4/2013 16Dept.of pharmaceutical analysis
  17. 17. 5/4/2013 17
  18. 18. These Rsmin are plotted as a function ofthe pH values for each column (Figure 3).The pH value with maximal Rsmin, the onewith the best separation for the worst-separated peak pair, is considered theoptimum on the considered column.For example, on column1, the pH withmaximal Rsmin is indicated on Figure 3. Thesame is done for all columns (Figure 3), andon each column the pH value with maximalRsmin is determined.Finally, the overall maximal Rsmin isdetermined. This overall maximal Rsminthen specifies the best pH and column. Forexample, on Figure 3, the pH with theoverall maximal Rsmin is indicated.5/4/2013 18Dept.of pharmaceutical analysis
  19. 19. 3.Optimization of the Organic Modifier CompositionModifier Mobile PhaseConcentrationApproximate pHTrifluoroacetic Acid (TFA) 0.05% 2Formic Acid 0.1% 2.8Ammonium Acetate + AceticAcid8 mM + 0.1% 4Ammonium Acetate 8 mM 7Ammonium Acetate +Ammonium Hydroxide8 mM + 0.05% 10.2Ammonium Hydroxide 0.05% 10.8Mobile Phase Modifiers10:5/4/2013 19Dept.of pharmaceutical analysis
  20. 20.  In general, for a three-component mixture, for eg. with modifiers methanol(MeOH), acetonitrile (ACN) and tetrahydrofurane (THF), all possiblecompositions can be represented in a triangle (Figure 1). Each vertex of the triangle represents a condition where the organic part ofthe mobile phase only consists of one modifier. Each side represents binary mixtures of organic modifiers and inside thetriangle ternary mixtures can be found. Additionally, each condition contains a given amount of water to make allcompositions isoeluotropic. Any composition can be considered as a mixture with given fractions of x1,x2 and x3, each ranging between 0 and 1. When applying a gradient elution, isoleluotropy of the beginning and endconditions often is ignored. In fact in a gradient elution one moves from one triangle to another, fromone with a larger water content to one with a smaller.5/4/2013 20Dept.of pharmaceutical analysis
  21. 21. Fig.1 Solvents triangle, with x1, for instance,representing an ACN/H2O mixture,x2 MeOH/H2O, and x3 THF/H2O. Watercontent is determined by solvent strength.Numbers 1–10 (see Table 1).5/4/2013 21Dept.of pharmaceutical analysis
  22. 22.  Here only one side of the triangle is explored (e.g., side 2 to 3 inFigure 1). Two different retention models can be constructeddepending on whether the model is based on two or threemeasurements (Figure 2).log(k) = b1x + b0 [1]log(k) = b11x2 + b1x + b0 [2]where k is the retention factor, x the fraction of one of both modifiers(between 0 and 100% or between 0 and 1) and b1, b0, b11, b1 andb0 are the regression coefficients of the models. To optimize a binary mixture of organic modifiers, the drug mixture isanalysed at two or three conditions and the model (Equations 1 or 2) isbuilt for each impurity.5/4/2013 22Dept.of pharmaceutical analysis
  23. 23.  In Figure 2, this is represented for four substances. For each intermediatemixture of organic modifiers, the retention is predicted for all impurities. At each composition, the retentions are then sorted and the selectivity factorα or the resolution Rs is calculated for each pair of consecutive peaks. To determine the optimal conditions, the minimal selectivity factor αmin orthe minimal resolution Rsmin is plotted as a function of the composition ofthe mobile phase. The plot does not have a smooth behavior because the plotted αmin orRsmin values at different conditions may originate from different peak pairs. That composition where αmin or Rsmin is maximal is then selected as theoptimum, for example X = 40% x3 in Figure 2(a), meaning that 2/5 ofcomposition x3 and 3/5 of composition x2 are mixed.5/4/2013 23Dept.of pharmaceutical analysis
  24. 24. Figure 2: Optimization of a binary mixture of organic modifiers using (a) a linear model, and(b) a quadratic polynomial model to model retention. Si = substance i from mixture.5/4/2013 24Dept.of pharmaceutical analysis
  25. 25. 4.Optimization of the Gradient Slope and the Temperature This step concerns factors with less influence on theselectivity. This step often can be considered optional or rather asa fine-tuning of the method. It can be done using response surface designs. In general, the optimized conditions for two examinedfactors, x1 and x2(e.g., gradient slope andtemperature), to separate a mixture of compounds, isderived from a plot of Rsmin values at different x1–x2grid conditions, as shown in Figure . Similar plots are obtained for the triangle (mixturedomain) when plotting Rsmin values as a function ofthe organic modifier composition5/4/2013 25Dept.of pharmaceutical analysis
  26. 26. Impurity profiling of Famotidine in bulk drugs and pharmaceuticalformulations by RP-HPLC method using ion pairing agent11 Column :C18(250 mm x 4.6 mm) Mobile phase :acetonitrile, methanol and 1-hexane sodium sulfonate. Flow rate : 1.5 ml/min. Detector photo diode array was operated at 266 nm. The degree of linearity of the calibration curves, the percent recoveries offamotidine and impurities, the limit of detection and quantitation, for the hplcmethod were determined. The method was found to be simple, specific, precise, accurate and reproducible.5/4/2013 26Dept.of pharmaceutical analysis
  27. 27. Fig.The chromatogram of Famotidine and its related impurities5/4/2013 27Dept.of pharmaceutical analysis
  28. 28. Numerous applications have been sought in the areas of drug designingand in monitoring quality, stability and safety of pharmaceutical compounds, whetherproduced synthetically, extracted from natural products or produced by recombinantmethods.Impurity profiling of API’s by RP-HPLC12Drug Impurities MethodCefdinir Related substances HPLCDonepezil Process related impurities HPLCLinezolid Process related impurities HPLCLoratidine Process related impurities HPLCRepaglinide Process related impurities HPLCRofecoxib Process related impurities HPLCZaleplon Process related impurities HPLC 28
  29. 29. Isolation and characterization of impurities isrequired for acquiring and evaluating data that establishesbiological safety which reveals the need and scope ofimpurity profiling of drugs in pharmaceuticalresearch.Orthogonal methods are necessary for ongoingassessment of method specificity5/4/2013 29Dept.of pharmaceutical analysis
  30. 30. 1. International Conference on Harmonisation of Technical Requirements for the Registration ofPharmaceuticals for Human Use (ICH) guideline Q3A(R2), Impurities in new drug substances, (2006). Pgno.1-112.Ahuja Satindar, Impurities evaluation of pharmaceuticals, Marcel & Dekker publication, Edition Second,New York :1998 PP1-17,95--1113. http://www.locumusa.com ,International Journal of Generic Drugs pgno.370 ,ISSN 0793 7784 Euro4. Method Development for Drug Impurity Profiling: Part 1,Apr 1, 2010,By: Bieke Dejaegher, Yvan VanderHeyden, Melanie Dumarey ,LCGC EUROPEVolume 23, Issue 4 ,chromatography.online.com5. E. Van Gyseghem et al., J. Chrom. A, 988, 77–93 (2003).6. E. Van Gyseghem et al., J. Pharm. Biomed. Anal., 41, 141–151 (2006).7. M. Dumarey et al., Anal. Chim. Acta, 609, 223–234 (2008).8. D. Visky et al., J. Chrom. A, 1101, 103–114 (2006).9.M. Dumarey et al., Anal. Chim. Acta, 656, 85–92 (2009).10.Development and Use of Orthogonal Methods for Impurity Profiling of Pharmaceuticals by HPLC byHenrik T. Rasmussen, Fengmei Zheng, Dora Visky, Rhonda Jackson, Analytical Development,slide 13-1411. Rewiew article:Impurity profiling of Famotidine in bulk drugs and pharmaceutical formulations byRP-HPLC method using ion pairing agent M.Vamsi Krishna*, G. Madhavi*, L. A. Rama Prasad**and D. Gowri Sankar**, Der Pharmacia Lettre, 2010,1-11(http://scholarsresearchlibrary.com/archive.html)12. Impurity profiling of pharmaceuticals, anita ayre et al:, ARPB, 2011; vol 1(2),Table:2,pg.No:865/4/2013 30Dept.of pharmaceutical analysis
  31. 31. 5/4/2013 31Dept.of pharmaceutical analysis
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