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Radiochemical Decomposition Carbon-14 labelled compounds Dr Sean L Kitson Almac Isotope Chemistry Laboratories [email_address] 14 C

Key Learning Objectives Identify and establish the likely optimum storage conditions for a particular ‘custom’ carbon-14 labelled compound Review the factors affecting the rate of decomposition of carbon-14 labelled compounds Describe some ways of minimizing the rates of decomposition

Beta Particles Ionizing radiation breaks chemical bonds and creates ions which damage surrounding tissue (matter) Beta emission is due to the excess number of neutrons in the nucleus 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

14 N 7 + 1 n 0 -> 14 C 6 + 1 H 1

Carbon-14: Martin Kamen 27-FEB-1940 T 1/2 = 5730 Years

Carbon-14 Radiotracer In pharmaceutical research carbon-14 is used as a tracer to ensure that potential drugs are metabolized without forming harmful by-products The carbon-14 label should ideally form part of the compound's molecular skeleton Used in biological research, agriculture, pollution control and archeology

Classes of Carbon-14 Labelled Compounds

Carbon-14 Custom Preparation Labelling Labelled building block (small molecule) then built up by multi-stage organic synthesis on multi- mg scale Purification Prep HPLC, Flash column chromatograhy, Crystallization Analysis HPLC, Radio-TLC, MS, NMR Dispensing Solid, Solution

Principle The compound itself and/or its immediate surroundings will absorb the radiation energy Energy absorbed by the compound will excite the molecules which can break-up or react with other molecules The excited decomposition fragments may also react with other labelled compounds producing other impurities 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

Mechanism of decomposition Fact: Compounds that are labelled with radioisotopes decompose faster than their unlabelled counterparts Consider: Half-life, Specific Activity (SA) and equally shelf-life of the labelled compound Most important is shelf-life -metabolic studies often require a radiochemical purity of at least 98%

Shelf-life The time during which a labelled compound may be used with confidence and safety, is important to both the user and the supplier The purity at which a radiolabelled compound ceases to be of use depends on the application

The Specific Activity (SA) of Compounds Specific Activity (SA) - the number of radioactive transformations per unit mass of a compound In general, the higher the SA of a compound, the greater the rate of decomposition This is constant for a given isotope in its atomic state and cannot be altered by chemical or environmental changes

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

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

Modes of Decomposition of Radiochemicals (1960)

Primary (internal) decomposition natural decay Results from the disintegration of the unstable nucleus of the radioactive atom Multiple carbon-14 label compounds on storage produce minute decomposition products eg 0.03% per year of [ 14 C]methylamine: 14 CH 3 14 CH 3 -> [ 14 CH 3 14 NH 3 ] -> 14 CH 3 NH 2 Yield of [1- 14 C]glycine from [2,3- 14 C]succinic acid is very small!

Primary (external) decomposition The beta particles (ionizing radiation) interacts with molecules of labelled compound surrounding the decaying nucleus Higher the SA , the greater the primary decomposition Can add unlabelled (carrier) or other solvent to reduce SA or increase the number of non-labelled molecules near each labelled molecule

Secondary decomposition 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 Difficult to control and is also the mode most susceptible to minor variations of the environmental conditions The low chemical weight of labelled compounds, particularly at high SA , intensifies the problems

Decomposition in aqueous solutions Many radiochemicals are soluble only in hydroxylic solvents and water is the ideal solvent! These include: amino acids, carbohydrates and nucleic acids Radiation chemistry of water is therefore an important consideration in the study of the stability of radiochemicals in aqueous systems Since, the effect on the labelled compound of the reactive species produced is the prime example of secondary decomposition

Ionizing radiation on water Primary entities believed to result from the radiolysis of water are: Hydronium ions Hydrated electrons Hydrogen atoms Hydroxyl radicals Hydroperoxy radicals Molecular hydrogen Hydrogen peroxide

In solution ionization occurs along the tracts of the beta particles in discrete pockets known as SPURS

Ionizing radiation on water Minimize decomposition -reducing the number of solute-radical interactions -Achieved by lowering the temperature of storage and by the use of radical scavengers -Free radical scavengers must react preferentially and rapidly with the reactive species present in solution with no further reaction - must terminate

Decomposition in organic solvents

Decomposition in organic solvents The detailed mechanism of self-decomposition of radiochemicals in organic solvents is unknown and is likely to be complex The transfer and absorption of the radiation energy is quite different and produces different forms of reactive species from those in aqueous systems

Organic Solvents Most widely used solvents include: -Benzene (sometimes) -Toluene -Ethanol -Methanol -Ethyl acetate -Pentane

Decomposition in organic solvents Chemical purity of the solvent is critical (use high purity solvents) Trace of peroxide in the solution may cause total destruction of the labelled compound (avoid diethyl ether, or other ethers) Chlorinated hydrocarbons (chloroform) must be avoided as solvents because of their potential quenching effect Chemical impurities may cause an increase in the rate of self-decomposition of the radiochemical on storage

Using Benzene Benzene provides a positive effect on other compounds Many sensitive radiochemicals at high SA for example steroids, fatty acids and hydrocarbons are remarkably stable towards self-radiolysis in benzene solutions 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

Steroids The addition of methanol to toluene/benzene solutions of labelled steroids has been observed to increase the rate of decomposition of these compounds Possibly by increasing the lifetime of polar attacking methoxy radicals Polar solvents such as methanol or water greatly accelerate the rate of self-decomposition of labelled steroids

Esters Acidic compounds containing carboxyl groups may become esterified on prolonged storage in alcoholic solution Formation of 24% [U- 14 C]phenylalanine ethyl ester on storage of [U- 14 C]phenylalanine in 95% ethanol at room temperature for 22 months

[ 14 C]-Amino acids (AA) At low SA (<30 mCi/mmol) AA are best stored as solids (freeze-dried) at 0 ° C or lower temperatures Under nitrogen, argon 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 If these conditions are followed the decomposition rate of [ 14 C]-amino acids does not exceed 2% per year Ethanol acts as a radical scavenger

Molecular Clustering At +2 ° C in the unfrozen state the solute molecules are free to move about in solution 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 This clustering of the solute molecules is much less marked in solutions that are frozen rapidly at -196 ° C (shelf-freezing)

Distribution of solute molecules (x) in aqueous and in frozen aqueous solutions – molecular clustering effect on slow freezing

Stability of [ 14 C]-Compound 100% 80% 90% Radiochemical purity 4 8 12 20 15 Time (weeks)

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

Effect of Temperature Stability of [ 14 C]-Compound 100% 70% 90% 80% -80º -20º 20º 6 3 1 Time (weeks) Radiochemical purity

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

‘ Ideal’ Radioactive Decomposition Rates

Screening Solvents: [ 14 C]-Compound

Principal guides that will help minimize decomposition Optimise storage conditions for chemical stability (correct pH, storage under inert gas etc ) As much as possible keep radiochemicals in the dark Store at low temperatures – solutions of radiochemicals should be stored cold but unfrozen (aqueous solutions at +2 ° C, ethanol solutions at -20 ° C) Compounds of low chemical stability should be stored at -80 ° C Compounds in their natural physical state should normally be stored at -20 ° C Dilute the SA – a compound at high SA will decompose faster than at lower SA Store as solutions – this effectively disperses the labelled molecules, decreasing the effect of secondary decomposition 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 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

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