ADVANCES IN PHARMACEUTICAL ANALYSIS Date : 24th March 2007 Presented by Manohar Sonanis General Manager (Analytical Research-API) Wockhardt Research Centre, Aurangabad 1

TECHNOLOGY OVERVIEW Instruments have revolutionized Chemical analysis. Replaces “Wet Chemistry” methods Provides higher sensitivity and accuracy. Increased lab productivity due to automation. Minimizes operators skill requirements and errors. 21 CFR, Part-11 compliance requirements. 2

NEW TECHNOLOGY INSTRUMENTS Separation Techniques High Performance Liquid Chromatography – HPLC,Thin Layer Chromatography Gas Chromatography, Ion Chromatography Mass Spectrometry LC MSMS,GC MS,Maldi,Time of Flight – ToF Life Science Instrumentation. Sequencing,DNA Synthesizer, Organic and Peptide Synthesis, Electrophoresis 3

NEW TECHNOLOGY INSTRUMENTS Spectroscopy Techniques. Atomic Absorption,UV and UV-Vis,ICP,ICP – MS,Fluorescence,Near Infrared X-Ray Fluorescence-Ray Diffraction, Inorganic Elemental Analysis, Surface Sciences Electron Microscopes, Light Microscopes, Scanning Material Characterization. Thermal Analysis,Viscometry ,PSD Lab Automation. Information Management,Robotics,Liquid Handling,Microplate Reading 4

DEMAND BY INDUSTRY 5

DEMAND BY FUNCTION IN PHARMA INDUSTRY 6

TECHNIQUES-APPLICATIONS HPLC : Separation of compounds(Qualitative&Quantitative) GC (Gas chromatography) : Analysis of volatile components LC-MS (High performance liquid chromatography hyphenated to Mass spectroscopy) : Impurity profiling. Clinical pharmacology. : Characterization PXRD (Powder x- ray diffractometer) : Polymorphs and Pseudo-polymorphs. : Patent protections. : Identification and Characterization DSC(Differential scanning calorimeter) : Drug development (Drug Excipient Interaction ). Polymorphism. 7

: Drug dissolution rate dependency. PSD (Particle size analyzer): Particle size determination of powder and suspension. : Drug dissolution rate dependency. FTIR (Fourier transform infra red spectroscopy) : Identification/characterization. Polymorphism. Others CE (Capillary : Separation of compounds electrophoresis) LCNMR : For impurity profiling NIR (Near infra : Characterization, polymorphs etc., red spectroscopy) Ion chromatography : Inorganic analysis (Cations & Anions) 8

HPLC (High Performance Liquid Chromatography) During 1970's, most chemical separations techniques were based on Open-column chromatography, Paper chromatography, and Thin-layer chromatography. Above techniques were inadequate in today's requirement Sensitivity, selectivity and reproducibility Quantification of compounds Resolution between similar compounds (Complex). 9

HPLC (High Performance Liquid Chromatography) Faster Analysis, Accurate quantification of compounds The continual refinements and innovations in HPLC instrument and column technologies have dramatic impacts on pharmaceutical analysis. The latest trends in HPLC analysis of small-molecule Drug Recent advances in instrumentation coupled with more consistent and efficient columns have increased the speed, sensitivity, and reliability of analytical methods. Cont… 10

HPLC (High Performance Liquid Chromatography) Principle Of HPLC Chromatographic separation process based on the difference in the surface interactions of the analyte and eluent molecules. 11

HPLC (High Performance Liquid Chromatography) UPLC 12

HPLC (High Performance Liquid Chromatography) HPLC Modes: Normal Phase: Polar stationary phase : Silica, Cyanopropyl Silyl, Phenyl, Amino Non-polar solvent : Dichloromethane, n-Hexane etc. Reverse Phase: Non-polar stationary : Octyl Silyl, Octadecyl Silyl phase Polar solvent : Methanol, Acetonitrile, Tetrahydrofuran, Water 13

HPLC (High Performance Liquid Chromatography) Columns: Solid Support : Backbone for bonded phases. Usually 10µ, 5µ or 3µ, 1.7µ silica or polymeric particles. Bonded Phase : Functional groups firmly linked (chemically bound) to the solid support, C-8, C18, C-4 etc. Extremely stable Reproducible Guard : Protects the analytical column Particles Interferences Prolongs the life of the analytical column Analytical : Performs the separation. 14

HPLC (High Performance Liquid Chromatography) Detectors UV Single wavelength (filter) Variable wavelength (monochromatic) Multiple wavelengths (PDA) Fluorescence Electrochemical Mass Spectrometry Refractive Index Evaporative light scattering Chiral 15

HPLC (High Performance Liquid Chromatography) 16

HPLC (High Performance Liquid Chromatography) 0.17% 17

Separation of 5 compounds in < 1 min. HPLC (High Performance Liquid Chromatography) Innovation of UPLC (Ultra High Pressure LC): Faster analysis Peak to peak resolution is improved High sensitivity and better S/N ratio Savings of time and cost per analysis Separation of 5 compounds in < 1 min. Fig. :UPLC stability indicating assay. Column: 2.1 _ 30 mm 1.7μm ACQUITY BEH C18 Peaks are in order : 5-nitroso-2,4,6-triaminopyrimidine 4-amino-6-chloro-1,3-benzenesulfanamide Hydrochlorthiazide Triamterine Methylbenzenesulfanamide 18

Identification Threshold3 Qualification Threshold3 HPLC (High Performance Liquid Chromatography) Impurity profiling in drug substances: Impurity Thresholds as per ICH (Q3A(R2)) Daily Dose1 Reporting Threshold2,3 Identification Threshold3 Qualification Threshold3 ≤ 2g/day 0.05% 0.10% or 1.0 mg per day intake (whichever is lower) 0.15% or 1.0 mg per day intake (whichever is lower) > 2g/day 0.03% Cont… 19

GC (Gas Chromatography) Packed column : Used for conventional low sensitive GC analysis Capillary Column : New technology columns used for better sensitivity generally with head space GC analysis. GC Tubing Column material Stationary phase Film thickness Inside diameter 20

GC (Gas Chromatography) Stationary Phases Polysiloxanes Low Bleed Phases (Arylene) Polyethylene Glycols 21

HS GC (Head Space GC Chromatogram) 22

Concentration limit (ppm) Toxic and environmental hazard GC (Gas Chromatography) ICH Q3C(R3): Impurities : Guideline for Residual Solvents TABLE 1. Class 1 solvents in pharmaceutical products (solvents that should be avoided). Solvent Concentration limit (ppm) Concern Benzene 2 Carcinogen Carbon tetrachloride 4 Toxic and environmental hazard 1,2-Dichloroethane 5 Toxic 1,1-Dichloroethene 8 1,1,1-Trichloroethane 1500 Environmental hazard 23

GC (Gas Chromatography) ICH Q3C(R3): Impurities : Guideline for Residual Solvents Class-2: solvents in pharmaceutical products to be limited. Class-3: Solvents with Low Toxic Potential (limit = 5000ppm) Class 3 Solvents Class 2 Solvents Acetic acid Heptane Acetonitrile Methanol Acetone Isobutyl acetate Chlorobenzene 2-Methoxyethanol Anisole Isopropyl acetate Chloroform Methylbutyl ketone 1-Butanol Methyl acetate Cyclohexane Methylcyclohexane 2-Butanol 3-Methyl-1-butanol 1,2-Dichloroethene N-Methylpyrrolidone Butyl acetate Methylethyl ketone Dichloromethane Nitromethane tert-Butylmethyl ether Methylisobutyl ketone 1,2-Dimethoxyethane Pyridine Cumene 2-Methyl-1-propanol N,N-Dimethylacetamide Sulfolane Dimethyl sulfoxide Pentane N,N-Dimethylformamide Tetralin Ethanol 1-Pentanol 1,4-Dioxane Toluene Ethyl acetate 1-Propanol 2-Ethoxyethanol 1,1,2-Trichloroethene Ethyl ether 2-Propanol Ethyleneglycol Xylene* Ethyl formate Propyl acetate Formamide Formic acid Tetrahydrofuran Hexane 24

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) Mass spectrometry instruments: Single Quad LCMS Tandem LC MSMS GC MS Maldi Time of Flight – ToF Introduction To LCMS Mass Spectrometry is superior scientific tool that provides greater specificity, sensitivity,and accuracy than conventional Ultraviolet or Fluorescent detectors. Mass Spectrometry is an analytical technique used for Identification of unknown compounds Quantification of known compounds Structural Elucidation 25

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) MASS Spectrometry: Measures the Masses of electrically charged molecules,or ions. Once ions are in the mass spectrometer they are sorted according to mass to charged ratio(m/z). A detector converts the signal to electrical signal , as a function m/z is converted by the data system into mass spectrum. 26

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) API 2000 Ion Path 27

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) Buffer and pH Control for LCMS Non-volatile buffers like phosphate are not recommended Use volatile buffers to replace phosphate such as Ammonium acetate, Formic acid, Acetic acid etc. Use a lower buffer concentrations:10-50mM LCMS Applications Identification of unknown impurities in Drug and its drug products. Identification of unknown impurities formed during degradation studies. To check the specificity of the HPLC Methods. Quantification of the substances which are very low in concentration and where it is difficult to use other techniques. 28

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) 29

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) 30

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) 31

LCMS (High Performance Liquid Chromatography Mass Spectroscopy) 32

ELSD (Light Scattering Evaporative Detector) Universal detector for HPLC Not dependant on the presence of chromophoric groups on the compound Analysis of semi volatile to nonvolatile compounds Typical mobile phase recommended as volatile buffers. Suitable for gradient applications. 33

XRD (X- Ray Powder Difractometer) In 1998, Abbott had to stop production of its HIV protease inhibitor, ritonavir (Norvir), because its manufacturing process was producing the wrong crystalline form of the compound. The new polymorphic structure of Norvir, one which had not been used in early testing of the drug, affected how the semisolid capsule dissolved. Patients were offered the drug in liquid form until the company could reformulate the product to eliminate the wrong structure. Glaxo Wellcome was in a lengthy court case against Novopharm for alleged patent infringement based on its right to manufacture and market a different polymorphic form of Glaxo’s best-selling anti-ulcer drug, Zantac. Glaxo’s patent on one polymorph of the drug expired in 1997, and Novopharm wanted to bring this form to the generic market. Glaxo argued that the generic form would infringe on its patent for the second crystalline form of the drug, the patent of which will not expire until 2002. The forms are therapeutically equivalent. Glaxo lost its case, and now Novopharm and other companies market generic Zantac for over-the-counter sales. 34

XRD (X- Ray Powder Difractometer) General principles of pharmaceutical solid polymorphism Importance of Pharmaceutical Solid Polymorphism Characterization of Polymorphs Influence of Polymorphism On Drug Substance And Drug Product Investigating the Polymorphs Setting Specifications for Polymorphs in Drug Substances Investigating the Importance of Setting Specifications for Polymorphs in Drug Products 35

XRD (Powder X- Ray Diffractometer) X-ray pattern of Amorphous compound X-ray pattern of crystalline Dihydrate compound X-ray powder diffraction pattern of the anhydrous and dihydrate compound. 36

XRD (Powder X- Ray Diffractometer) Quantification of Polymorph into another morph Fig-A Fig-B Powder diffraction patterns of the mixtures of Anhydrous and crystalline dihydrate molecules (in the amounts 0% (sample), 0.1%, 0.2%, 0.3%, 0.5% of the dihydrate form) Powder diffraction patterns of the mixtures of Anhydrous and crystalline dihydrate (in the amounts 0% (sample) and 4.0% with placebo in Tablet). 37

XRD (Powder X- Ray Diffractometer) Quantification of Polymorph into another morph 38

DSC (Differential Scanning Calorimeter) Applications of Thermal Analysis in Pharmaceuticals To measure physical and energetic properties with precision and accuracy Thermal analysis is used in the following areas Determination of purity Melting point, Polymorphism, Glass transition temperature, Drug- excipient interactions, Thermal stability To compare the results for the individual components and the mixture 39

DSC (Differential Scanning Calorimeter) DSC of Monohydrate DSC of Anhydrous 40

DSC (Differential Scanning Calorimeter) DSC of Monohydrate + Anhydrous 1:1 Monohydrate Characteristic onset 41

DSC (Differential Scanning Calorimeter) Quantification of Monohydrate in Anhydrous Drug Substance 42

CONCLUSION Technological advances are being happened in pharmaceutical analysis as like other areas of science. Due to technological advances more and more pure drug is available and will be available for patients and drug testing becomes more regulated. With the new technology instruments and automation productivity is dramatically increased and hence it benefits the business of the company. Indian pharma companies are more competent to the global market. With the support of quality analytical data companies are able to protect there innovations by patenting them. 43

Thank You 44

ANY QUESTIONS ?