Pharmaceutical Bioanalytics (2012-2013)

1. Techniques used in sample preparation (SPE, SPME, HS, LPME, TFC). 2. Accelerated solvent extraction techniques (SFE, ASE, MAE). Atmospheric pressure and high pressure digestion (vet and dry ashing, bomb digestion). 3. The origin of molecular spectra, characteristics of absorption spectrum, spectral effects. Construction UV-VIS spectrophotometers, cuvettes, application of UV-VIS spectrophotometry. 4. The origin of IR spectrum (vibrations), characteristics of IR spectrum, relationship between wavelength and wave number. Application of IR spectroscopy in the identification of organic compounds (characteristic group frequencies). 5. Main groups of chromatographic techniques (methods of development, technical settings, grouping by phases, separating chromatogram (tR, tm, VR, Vm, R, H, N). 6. Instrumental separating techniques (GC, HPLC), construction of the instruments, applications. 7. Constructions of MS instruments (ion sources and analyzers). 8. Characteristics of MS spectra, basic rules in MS (nitrogen rule, US), ionic patterns in MS, fragmentation reactions. Application of MS in the identification of organic compounds. 9. Hyphenated methods (GC-MS, LC-MS, MS-MS), construction of instruments (interfaces), applications. 10. Human drug metabolism (microsomal and non-microsomal oxidation, CYP 450 enzymes, conjugation). 11. In vitro and ex vivo techniques used in drug metabolism studies. Biomimetic model systems of human drug metabolism. 12. What kind of effects of the protein stability do you know, that we need to consider in protein isolation and purification? 13. Homogenization of biological samples. 14. Isolation of protein and total RNA from human tissue. 15. What do we need to pay attention to, and why in the RNA isolation? What kind of RNA isolation methods do you know? procedures), characteristics of a

16. What kind of DNA isolation methods do you know? 17. Preparing cDNA by Reverse Transcription PCR reaction. 18. Principles of PCR and application of PCR. 19. The main steps of PCR reaction and characterization of a basic PCR set up components and reagents. 20. Characterization of the Reverse Transcription PCR, primers and the usage of RT-PCR. 21. Agarose Gel Elektrophoresis of PCR products. 22. What is the basic of Real-Time PCR? List, group and characterize the Real-Time PCR probes! How do they work? 23. What is the difference between the end-point and the Real-Time PCR? Characterize the Real-Time PCR amplification curve! What are the advantages of the kinetic/RealTime PCR? Potential applications. 24. What is melting point? What is the basic of the melting-point analysis? What is good for? Draw and characterize a melting curve. What kind of quantification methods do you know? (Only list and group them!) 25. Principles of Western blot? Visualization techniques. Basic, electrophoresis (SDSPAGE), transfer step (electro blot, membrane types), blocking (BSA, non fat milk). 26. Direct detection/satining, indirect detection/staining. Comparison of monoclonal antibody/ polyclonal antibody. 27. Direct and indirect detection methods used in immunohistochemistry. The main characteristics of monoclonal and polyclonal antibodies. 28. Northern blot and its application. Southern blot and its application. 29. Principles of receptor pharmacology. 30. Saturation and displacement analysis as major receptor analitical tools.. 31. Immunoassay methods generally. 32. EIA, ELISA, FIA, FPIA, DELFIA 33. LIA, ECL, MIA, IEP, IP, Immunchomatography 34. RIA and design of RIA measurement. 35. Compare the main characteristics of Immunohistochemistry and Fluorescence in situ Hybridization 36. List and characterize the main steps of immunohistochemistry. Give examples for its application. Tissue microarray.

37. Introduce DNA sequencing through the description of one of the following methods: Maxam-Gilbert Sequencing; Sanger Chain Termination Method; Sequencing by Ligation (2-Base Encoding).