Solid-state analysis in pharmaceutical industry Tomáš Pekárek Tomas.Pekarek@zentiva.com Topics Overview • Generics • Drug Discovery and Development • Legal Aspects • Solid forms • Electromagnetic Spectrum • Microscopy • Particles size distribution • Infrared spectroscopy • Raman spectroscopy • Mapping • Thermal analysis 2 Differences between Original and Generics Analysis None Generics contain the same type of compounds like the originals Generics are also the drug product The goal is the same for generic and innovative drugs – safety and efficacy Reverse engineering – as much information on the original drug product as possible 3 Allotropy The property of chemical elements to exist in more than one crystalline lattice Examples: graphite vs. Diamond vs. Other forms 4 Autor: Anton – Na Commons přeneseno z de.wikipedia.; original upload 7. Feb 2004 by Anton, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=350220 https://cs.wikipedia.org/wiki/Uhl%C3%ADk Allotropy Phosphorus white and red (and others) 5 Volné dílo, https://commons.wikimedia.org/w/index.php?curid=1311089 Autor: Ondřej Mangl – ChemSketch, Volné dílo, https://commons.wikimedia.org/w/index.php?curid=2135934 Polymorphism 6 http://chemistry.tutorvista.com/inorganic- chemistry/polymorphism.html http://www.physics-in-a-nutshell.com/article/1 Crystalline - amorphous Polymorphism – The property of chemical compounds to exist in more than one crystalline lattice. Different polymorphs – Different properties similarly to allotropes: density, hardness, reactivity, dissolution rate Amorfy – bez pravidelné struktury Solid State Analytical Techniques – the most commonly used methods in pharmaceutical practise Microscopy and particle size Analysis • Optical • Electron • Image Analysis • … Thermal Analysis • Differential Scanning Calorimetry (DSC) • Thermogravimetric Analysis (TGA) • Dynamic Vapour Sorption (DVS) • … X-ray diffraction • Single- crystal • Powder diffraction Molecular spectroscopy • Infrared • Raman • Nuclear Magnetic Resonance (NMR) • Mass Spectroscopy • … 7 Electromagnetic Spectrum 8 By Inductiveload, NASA - self-made, information by NASABased off of File:EM Spectrum3new.jpg by NASAThe butterfly icon is from the P icon set, File:P biology.svgThe humans are from the Pioneer plaque, File:Human.svgThe buildings are the Petronas towers and the Empire State Buildings, both from File:Skyscrapercompare.svg, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2974242 Infrared Spectroscopy http://www.lightsources.org/imagebank/image/electromagnetic-spectrum 9 http://www2.lbl.gov/MicroWorlds/ALSTool/EMSpec/EMSpec2.html Electromagnetic Spectrum Infrared Region 10 Wavelength μm Wavenumber cm-1 Region 0.78-2.5 12800-400 Near infrared (NIR) 2.5-40 4000-200 Mid infrared (MIR) 40-1000 200-5 Far infrared (FIR) Infrared Spectroscopy • Onephotonic transition between two vibrational levels • Interaction with the incidental photon • Mid IR Region (MIR) – the most universal method in development and QC • Near IR Region (NIR) – mainly in QC • Far IR (FIR) – mainly in primary research 11 Infrared Spectroscopy 3N-6 (3N-5 for linear molecules) degrees of freedom (vibrational modes): 3 degrees of freedom for one atom (independent translation – 3 coordinates) For N-atomic molecule 3 x N = 3N degrees of freedom (translation, rotation, vibration) For IR vibration 3 rotations (2 for linear molecule) and 3 translation doesn‘t count, thus: 3N-6 degrees of freedom for non-linear molecule vibrations 12 Infrared Spectroscopy 13 Dipole moment change condition , where q is a normal coordinate Stretching vibrations – the bond length changes Symetric Antisymetric www.wikipedia.org Infrared Spectroscopy 14 www.wikipedia.org Deformation and bending modes – bonding angle changes Twisting Wagging Rocking Scissoring Infrared Spectroscopy Because of dipole change condition, homoatomic molecules do not absorb IR radiation (O2, N2 , H2 , …) Molecules exhibiting dipole moment absorb usually strongly, because the dipole change is high (C=O, S=O, N=O, O-H, …) (Dis)advantage for pharmaceutical analysis, as formulations often contain saccharides containing many hydroxyl groups 15 Infrared Spectroscopy – Instrumentation Source: MIR: ceramic rod T 1000-1200 °C NIR: halogen nebo tungsten bulb FIR: mercury lamp Detector: MIR: deuterated triglycin sulphate (DTGS), mercury-cadmium tellurid (MCT) NIR: PbSe, PbS, Ge, MCT FIR: DTGS Optical elements must be IR stransparent: KBr, ZnSe, CaF2, CsI, Si, diamond, … 16 Interference 17 http://www.ctgclean.com/tech- blog/2011/10/ultrasonics-sound- interactions-of-sound-waves/ https://www.ligo.caltech.edu/LA/page/what-is-interferometer Infrared Spectroscopy - Interferometer 18 Fixed Mirror Moving Mirror IR Source Detector Sample Beam-splitter Infrared spectroscopy – measurement techniques Transmission • Solids, liquids nad gasses incl. emulsions and suspensions Reflective • Liquids, solids, emulsions and suspensions Fiber Techniques Microscopic techniques 19 Infrared spectroscopy – Transmission Transmission: Cuvettes – for liquids and gasses and suspensions of solids (Nujol, Fluorolube) • Transparent cuvettes – KBr, CaF2, BaF2, KRS-5 (TlBr-TlI) • Resistance to the analyzed material Discs – with potassium bromide • Possible polymorph changes • Possible interaction with groups such as: –NHx, -COOH, … 20 Electromagnetic Spectrum – Effects Diffraction and refraction: Reflection: Same medium => Same speed, Wavelength Refraction: Different medium => Different Wavelengths Diffraction: Same medium => Wavelengths differentiation Reflection: 21 By Cmglee - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=19051904 By Zátonyi Sándor (ifj.) - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=10035697 Infrared spectroscopy – Reflection Attenuated Total Reflection (ATR) 22 Evanescent wave IRE (ATR crystal) Sample Incidental beamReflected beam Infrared spectroscopy – Reflection Attenuated Total Reflection (ATR): Tight contact of the IRE and the sample is necessary Critical Angle (reflection only, not the refraction): Depth of penetration: θ angle of incidental beam, n2 refractive index of the sample, n1 refractive index of IRE 23 Infrared spectroscopy – Identity confirmation 24 Infrared spectroscopy – Bands assignment 25 3000-2600 cm-1 broad band ν(O-H), 3100 – 3000 cm-1 ν(C-H) aromatic, 3000-2800 cm-1 ν(C-H) aliphatic, 1754 cm-1 ν(C=O) esther, 1692 cm-1 ν(C=O) acid, 1606 cm-1 ν(C=C) aromatic, 1458 δ(CH3), 1221 cm-1 νas(C-O), 1188 cm-1 νsym(C-O). Infrared spectroscopy – Polymorph differentiation 26 Form I Form II Infrared spectroscopy – Salt differentiation 27 Raman Spectroscopy Sir Chandrasekhara Venkata Raman (1888-1970) • Nobel Prize 1930 Raman Effect (together with Sir Kariamanickam Srinivasa Krishnan) - 28 February 1928 • Non-elastic scattering in liquids • Published sooner than Grigory Samuilovich Landsberg and Leonid Isaakovich Mandelstam - 21 February 1928 • Soviet Union • In crystals Predicted by Austrian Adolf Gustav Stephan Smekal (1923) 28 Raman Spectroscopy 29 Basic electronic state Excited electronic state Virtual levels Fluorescence Anti- Stokes StokesRayleighIR 3 4 1 2 5 ν = 3 ν = 2 ν = 1 ν = 0 ν – vibrational quantum number 1 – absorbed photon (ν0) 2 – scatered photon (ν0) 3 – scatered photon (ν0 - νvib) 4 – scatered photon (ν0 + νvib) 5 – fluorescence Raman Spectroscopy Comparison with IR spectroscopy: Infrared: one-photonic effect (absorption) Principle: Intensity: Raman: Two-photonic effect (non-elastic scattering) Principle: Intensity: Identical vibrations – different intensity 30 IR vs. Raman Spectroscopy 31 MIR RS OH CH C=CC=O Raman Spectroscopy Instrumentation: Source: • Lasers (monochromatic, coherent – identical frequency, direction, phase of photons • NIR Nd:YAG (1064 nm), diode (785 nm), … • VIS Nd:YAG (532 nm), He-Ne (633), Ar (514, 488, 458), … • UV Ar (244, 257), HeCd (325), … • Mercury lamp Detectors • Ge, CCD, photomultipliers, .. 32 Electromagnetic Spectrum – Laser Light Amplification by Stimulated Emission of Radiation 33 1 – medium, 2 – energy pump, 3 – high reflection mirror, 4 – partially transparent mirror, 5 – laser beam By User:Tatoute - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=577575 Raman Spectroscopy Dispersive elements • Gratings • Lower accumulation time • Better signal to noise • Higher risk of sample burning • UV and VIS lasers Interferometric systems • Lower risk of fluorescence • Higher acqusition time • Lower spectra intensity • NIR lasers 34 Fixed Mirror Moving Mirror Source / Sample Detector Sample Beam- splitter Raman Spectroscopy 35 P. Matějka, přednášky VŠCHT Praha Raman Spectroscopy 36 formT17 formA formB 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 Int 6008001000120014001600 Wavenumbers(cm-1) Raman Spectroscopy – Application in pharmaceutical industry Same as IR plus: Possible to measure through glass and plastic packages (no contamination risk) Possible to measure aqueous samples – low OH band intensity No or minimal sample pretreatment Intensive symetric vibrations (C=C, N=N, …) – advantage for API identification in the mixtures and/or dosage form Possible to calculate the temperature of the sample 37 Microscopy – Sample preparation Many approaches Consider what you want to achieve Directly on a glassy carrier, Petri dish, … Sectioning is carried out by a knive, blades, abrasive paper, … For thin sections of hard, soft or small materials: embedding into a matrix and consequent sectioning or abrasion Embedding is appropriate among others for mineralogy, restoration, histology, tissue engineering and pharmaceutics 38 Microscopy – Sample preparation Sample is embedded into an appropriate medium blocks (parraffin, resins, polyurethans, …) 39 https://www.leicabiosystems.com/ http://sites.gsu.edu/neuroscience-core/training/histology/ Microscopy – Sample preparation Blocks are consequently abraded or sectioned by microtome 40 https://www.leicabiosystems.com/ Raman Spectroscopy – microscopy, confocality 41 http://fb6www.uni-paderborn.de/.../microscope_e.htm Raman spectroscopy – mapping Thermal, IR, Raman 42 Raman spectroscopy – mapping Thermal, IR, Raman 43 Raman spectroscopy – mapping Mapping Thermal, IR, Raman 44 Raman spectroscopy – mapping 45 Raman spectroscopy – mapping 46 Raman Spectroscopy – Probes Solid form transformation 47 Raman Spectroscopy: Other Applications ● All research and development fields, e.g.: ● Medicine – tissues diagnostics ● Geology – mineral analysis ● Electrotechnics – semiconductor applications, microchips ● Chemical industry ● Food-Processing industry ● Pharmaceutical industry ● Metallurgy ● Rescue Systems ● Armed Forces & criminalistics & Forensic Sciences 48 Thermal Analysis Physico-chemical properties are observed • Changes of mass, energy, conductivity, etc. during heating • Different principles 49 Thermogravimetry (TGA) Accurate balances Weight change during heating Application: • Water / moisture content • Thermal stability • Thermal decomposition • Weight changes 50 http://radchem.nevada.edu/classe s/chem455/lecture_22__thermal_ methods.htm Thermogravimetry (TGA) 51 Water-free vs. 7 mol water Differential Scanning Calorimetry (DSC) Method • Amount of supplied energy in order to keep isothermal conditions (heated sample and reference) Application: • Melting point • Glassy transition temperature • Phase transitions • Polymorphism • Thermal decomposition • Dehydratation References: • DSC – empty cup • MicroDSC – reference: according to specific thermal capacity: corundum, water and undekan (90 % of measurement) 52 http://www.intechopen.com/books/applications-of-calorimetry-in-a-wide-context-differential-scanning-calorimetry-isothermal- titration-calorimetry-and-microcalorimetry/thermal-analysis-of-phase-transitions-and-crystallization-in-polymeric-fibers Differential Scanning Calorimetry (DSC) 53 Crystalline Amorphous Dynamic Vapour Sorption Hygroscopicity • A potential of substances to sorb and retain water • Affects physico-chemical properties and material stability • Registration of weight changes depending on the controlled relative humidity, time and temperature 54 Dynamic Vapour Sorption Adsorption – surface interaction, no penetration into the bulk Absorption – penetration of water into the bulk 55 http://dunia-wahyu.blogspot.cz/2012/06/dynamic-vapour- sorption-dvs-analysis.html Dynamic Vapour Sorption 56 By Dvstechnique - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=7321903 Surface Area Analysis Adsorption of gas (helium or nitrogen) or mercury on the material surface Environment: T = -196 °C (liquid nitrogen boiling point) Non-covalent interactions (van deer Waals), between gas and surface Sample preparation – degas (vacuum) Higher surface = larger interaction area 57 Surface Area Analysis 58 http://www.quantachrome.com/gassorption/nova_series.html Surface Area Analysis 59 5m2/g 10m2/g 150m2/g Thank you