Stability Of Carbon 14 Labelled Compounds

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A presentation on the stability of carbon-14 labelled compounds

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Stability Of Carbon 14 Labelled Compounds

  1. 1. Radiochemical Decomposition Carbon-14 labelled compounds Dr Sean L Kitson Almac Isotope Chemistry Laboratories [email_address] 14 C
  2. 2. Key Learning Objectives <ul><li>Identify and establish the likely optimum storage conditions for a particular ‘custom’ carbon-14 labelled compound </li></ul><ul><li>Review the factors affecting the rate of decomposition of carbon-14 labelled compounds </li></ul><ul><li>Describe some ways of minimizing the rates of decomposition </li></ul>
  3. 3. Types of Radiation
  4. 5. Beta Particles <ul><li>Ionizing radiation </li></ul><ul><li>breaks chemical bonds and creates ions which damage surrounding tissue (matter) </li></ul><ul><li>Beta emission is due to the excess number of neutrons in the nucleus </li></ul><ul><li>When there are significantly more neutrons than protons in a nucleus, the neutrons degenerate into protons and electrons, which are ejected from the nucleus at high velocities </li></ul>
  5. 7. 14 N 7 + 1 n 0 -> 14 C 6 + 1 H 1
  6. 8. Radioisotopes
  7. 9. Carbon-14: Martin Kamen 27-FEB-1940 T 1/2 = 5730 Years
  8. 10. Carbon-14 Radiotracer <ul><li>In pharmaceutical research carbon-14 is used as a tracer to ensure that potential drugs are metabolized without forming harmful by-products </li></ul><ul><li>The carbon-14 label should ideally form part of the compound's molecular skeleton </li></ul><ul><li>Used in biological research, agriculture, pollution control and archeology </li></ul>
  9. 11. Classes of Carbon-14 Labelled Compounds
  10. 12. Carbon-14 labelled Drugs
  11. 13. Carbon-14 Building Blocks
  12. 14. Carbon-14 Custom Preparation <ul><li>Labelling </li></ul><ul><ul><li>Labelled building block (small molecule) then built up by multi-stage organic synthesis on multi- mg scale </li></ul></ul><ul><li>Purification </li></ul><ul><ul><li>Prep HPLC, Flash column chromatograhy, Crystallization </li></ul></ul><ul><li>Analysis </li></ul><ul><ul><li>HPLC, Radio-TLC, MS, NMR </li></ul></ul><ul><li>Dispensing </li></ul><ul><ul><li>Solid, Solution </li></ul></ul>
  13. 15. Radio-decomposition
  14. 16. Principle <ul><li>The compound itself and/or its immediate surroundings will absorb the radiation energy </li></ul><ul><li>Energy absorbed by the compound will excite the molecules which can break-up or react with other molecules </li></ul><ul><li>The excited decomposition fragments may also react with other labelled compounds producing other impurities </li></ul><ul><li>Energy absorbed by the immediate surrounding (often the solvent) can produce reactive species such as free radicals which can result in the destruction of the labelled compound </li></ul>
  15. 17. Mechanism of decomposition <ul><li>Fact: Compounds that are labelled with radioisotopes decompose faster than their unlabelled counterparts </li></ul><ul><li>Consider: Half-life, Specific Activity (SA) and equally shelf-life of the labelled compound </li></ul><ul><li>Most important is shelf-life </li></ul><ul><li>-metabolic studies often require a radiochemical purity of at least 98% </li></ul>
  16. 18. Shelf-life <ul><li>The time during which a labelled compound may be used with confidence and safety, is important to both the user and the supplier </li></ul><ul><li>The purity at which a radiolabelled compound ceases to be of use depends on the application </li></ul>
  17. 19. The Specific Activity (SA) of Compounds <ul><li>Specific Activity (SA) - the number of radioactive transformations per unit mass of a compound </li></ul><ul><li>In general, the higher the SA of a compound, the greater the rate of decomposition </li></ul><ul><li>This is constant for a given isotope in its atomic state and cannot be altered by chemical or environmental changes </li></ul>
  18. 20. Specific Activity (SA) of molecules C SA = 62.4 mCi/mmol C C C C C C [1- 14 C] compound SA = 62.4 mCi/mmol C C C C C C [U- 14 C] compound SA= 6 x 62.4 = 374.4 mCi/mmol
  19. 21. Decreasing the Specific Activity (SA) Adjusted SA = 62.4/6 = 10.4 mCi/mmol Add 5 equivalents of inactive compound C C C C C C [1- 14 C] compound SA = 62.4 mCi/mmol C C C C C C
  20. 22. Modes of Decomposition of Radiochemicals (1960)
  21. 23. Primary (internal) decomposition natural decay <ul><li>Results from the disintegration of the unstable nucleus of the radioactive atom </li></ul><ul><li>Multiple carbon-14 label compounds on storage produce minute decomposition products </li></ul><ul><li>eg 0.03% per year of [ 14 C]methylamine: </li></ul><ul><li>14 CH 3 14 CH 3 -> [ 14 CH 3 14 NH 3 ] -> 14 CH 3 NH 2 </li></ul><ul><li>Yield of [1- 14 C]glycine from [2,3- 14 C]succinic acid is very small! </li></ul>
  22. 24. Primary (external) decomposition <ul><li>The beta particles (ionizing radiation) interacts with molecules of labelled compound surrounding the decaying nucleus </li></ul><ul><li>Higher the SA , the greater the primary decomposition </li></ul><ul><li>Can add unlabelled (carrier) or other solvent to reduce SA or increase the number of non-labelled molecules near each labelled molecule </li></ul>
  23. 25. Carbon-14 Compounds
  24. 26. Secondary decomposition <ul><li>This type of decomposition is the most damaging mode and results from the interaction of labelled molecules - free radicals or other excited species produced by the radiation </li></ul><ul><li>Difficult to control and is also the mode most susceptible to minor variations of the environmental conditions </li></ul><ul><li>The low chemical weight of labelled compounds, particularly at high SA , intensifies the problems </li></ul>
  25. 27. Decomposition in aqueous solutions <ul><li>Many radiochemicals are soluble only in hydroxylic solvents and water is the ideal solvent! </li></ul><ul><li>These include: amino acids, carbohydrates and nucleic acids </li></ul><ul><li>Radiation chemistry of water is therefore an important consideration in the study of the stability of radiochemicals in aqueous systems </li></ul><ul><li>Since, the effect on the labelled compound of the reactive species produced is the prime example of secondary decomposition </li></ul>
  26. 28. Ionizing radiation on water <ul><li>Primary entities believed to result from the radiolysis of water are: </li></ul><ul><li>Hydronium ions </li></ul><ul><li>Hydrated electrons </li></ul><ul><li>Hydrogen atoms </li></ul><ul><li>Hydroxyl radicals </li></ul><ul><li>Hydroperoxy radicals </li></ul><ul><li>Molecular hydrogen </li></ul><ul><li>Hydrogen peroxide </li></ul>
  27. 29. In solution ionization occurs along the tracts of the beta particles in discrete pockets known as SPURS
  28. 30. Ionizing radiation on water <ul><li>Minimize decomposition </li></ul><ul><li>-reducing the number of solute-radical interactions </li></ul><ul><li>-Achieved by lowering the temperature of storage and by the use of radical scavengers </li></ul><ul><li>-Free radical scavengers must react preferentially and rapidly with the reactive species present in solution with no further reaction - must terminate </li></ul>
  29. 31. Decomposition in organic solvents
  30. 32. Decomposition in organic solvents <ul><li>The detailed mechanism of self-decomposition of radiochemicals in organic solvents is unknown and is likely to be complex </li></ul><ul><li>The transfer and absorption of the radiation energy is quite different and produces different forms of reactive species from those in aqueous systems </li></ul>
  31. 33. Organic Solvents <ul><li>Most widely used solvents include: </li></ul><ul><li>-Benzene (sometimes) </li></ul><ul><li>-Toluene </li></ul><ul><li>-Ethanol </li></ul><ul><li>-Methanol </li></ul><ul><li>-Ethyl acetate </li></ul><ul><li>-Pentane </li></ul>
  32. 34. Decomposition in organic solvents <ul><li>Chemical purity of the solvent is critical (use high purity solvents) </li></ul><ul><li>Trace of peroxide in the solution may cause total destruction of the labelled compound (avoid diethyl ether, or other ethers) </li></ul><ul><li>Chlorinated hydrocarbons (chloroform) must be avoided as solvents because of their potential quenching effect </li></ul><ul><li>Chemical impurities may cause an increase in the rate of self-decomposition of the radiochemical on storage </li></ul>
  33. 35. Using Benzene <ul><li>Benzene provides a positive effect on other compounds </li></ul><ul><li>Many sensitive radiochemicals at high SA for example steroids, fatty acids and hydrocarbons are remarkably stable towards self-radiolysis in benzene solutions </li></ul><ul><li>The addition of a secondary solvent to benzene solution of a radiochemical, normally added to increase the solubility of the compound in the solvent mixture, sometimes leads to an acceleration of self-decomposition </li></ul>
  34. 36. Steroids <ul><li>The addition of methanol to toluene/benzene solutions of labelled steroids has been observed to increase the rate of decomposition of these compounds </li></ul><ul><li>Possibly by increasing the lifetime of polar attacking methoxy radicals </li></ul><ul><li>Polar solvents such as methanol or water greatly accelerate the rate of self-decomposition of labelled steroids </li></ul>
  35. 37. Esters <ul><li>Acidic compounds containing carboxyl groups may become esterified on prolonged storage in alcoholic solution </li></ul><ul><li>Formation of 24% [U- 14 C]phenylalanine ethyl ester on storage of </li></ul><ul><li>[U- 14 C]phenylalanine in 95% ethanol at room temperature for 22 months </li></ul>
  36. 38. [ 14 C]-Amino acids (AA) <ul><li>At low SA (<30 mCi/mmol) AA are best stored as solids (freeze-dried) at 0 ° C or lower temperatures </li></ul><ul><li>Under nitrogen, argon </li></ul><ul><li>At higher SA best stored in aqueous solutions containing 2% ethanol at a radioactive concentration of 50-100 uCi/ml at +2 ° C or frozen at -20 ° C or lower temperature </li></ul><ul><li>If these conditions are followed the decomposition rate of [ 14 C]-amino acids does not exceed 2% per year </li></ul><ul><li>Ethanol acts as a radical scavenger </li></ul>
  37. 39. Molecular Clustering <ul><li>At +2 ° C in the unfrozen state the solute molecules are free to move about in solution </li></ul><ul><li>In solutions that are frozen slowly, say at -20 ° C to -40 ° C, molecular clustering of the solute occurs as freezing of pure solvent around the edge of the sample occurs first </li></ul><ul><li>This clustering of the solute molecules is much less marked in solutions that are frozen rapidly at -196 ° C (shelf-freezing) </li></ul>
  38. 40. Distribution of solute molecules (x) in aqueous and in frozen aqueous solutions – molecular clustering effect on slow freezing
  39. 41. Stability of [ 14 C]-Compound 100% 80% 90% Radiochemical purity 4 8 12 20 15 Time (weeks)
  40. 42. Effect of Specific Activity Decomposition of [ 14 C]-Compound at 20°C 100% 30% 90% 60% 20 55 120 Specific activities in mCi/mmol 7 Time (days) Radiochemical purity
  41. 43. Effect of Temperature Stability of [ 14 C]-Compound 100% 70% 90% 80% -80º -20º 20º 6 3 1 Time (weeks) Radiochemical purity
  42. 44. Effect of Free Radical Scavengers Decomposition of [ 14 C]-Compound at 20ºC 100% 90% 80% 70% 4 2 3 1 Time (months) Radiochemical purity + 3% ethanol Aqueous solution
  43. 45. ‘ Ideal’ Radioactive Decomposition Rates
  44. 46. Screening Solvents: [ 14 C]-Compound
  45. 47. Principal guides that will help minimize decomposition <ul><li>Optimise storage conditions for chemical stability (correct pH, storage under inert gas etc ) </li></ul><ul><li>As much as possible keep radiochemicals in the dark </li></ul><ul><li>Store at low temperatures – solutions of radiochemicals should be stored cold but unfrozen (aqueous solutions at +2 ° C, ethanol solutions at </li></ul><ul><li>-20 ° C) </li></ul><ul><li>Compounds of low chemical stability should be stored at -80 ° C </li></ul><ul><li>Compounds in their natural physical state should normally be stored at </li></ul><ul><li>-20 ° C </li></ul><ul><li>Dilute the SA – a compound at high SA will decompose faster than at lower SA </li></ul><ul><li>Store as solutions – this effectively disperses the labelled molecules, decreasing the effect of secondary decomposition </li></ul><ul><li>Add radical scavengers or other stabilizers –when compatible with the use, adding a radical scavenger (2-3% ethanol added to an aqueous solution) can lead to an increased shelf life </li></ul><ul><li>Avoid reopening of vials, and warming/cooling cycle – if a radiochemical is to be used over several weeks/months, it is best to have it sub-aliquot in a number of vials, keeping those to be used later in the refrigerator or freezer until required </li></ul>

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