RadiochemicalRadiochemical Decomposition DecompositionCarbon-14 labelled compoundsCarbon-14 labelled compounds

Dr Sean L KitsonDr Sean L KitsonAlmac Isotope Chemistry LaboratoriesAlmac Isotope Chemistry Laboratories

Sean.kitson@almacgroup.comSean.kitson@almacgroup.com

1414CC

Key Learning ObjectivesKey Learning Objectives

Identify and establish the likely optimum Identify and establish the likely optimum storage conditions for a particular ‘custom’ storage conditions for a particular ‘custom’ carbon-14 labelled compoundcarbon-14 labelled compound

Review the factors affecting the rate of Review the factors affecting the rate of decomposition of carbon-14 labelled decomposition of carbon-14 labelled compoundscompounds

Describe some ways of minimizing the Describe some ways of minimizing the rates of decompositionrates of decomposition

Types of RadiationTypes of Radiation

Beta ParticlesBeta Particles Ionizing radiationIonizing radiation breaks chemical bonds and creates ions which breaks chemical bonds and creates ions which

damage surrounding tissue (matter)damage surrounding tissue (matter) Beta emission is due to the excess number of Beta emission is due to the excess number of

neutrons in the nucleusneutrons in the nucleus When there are significantly more neutrons When there are significantly more neutrons

than protons in a nucleus, the neutrons than protons in a nucleus, the neutrons degenerate into protons and electrons, which degenerate into protons and electrons, which are ejected from the nucleus at high velocitiesare ejected from the nucleus at high velocities

14N7 + 1n0 → 14C6 + 1H1

RadioisotopesRadioisotopes

Carbon-14: Martin KamenCarbon-14: Martin Kamen 27-FEB-1940 27-FEB-1940

TT1/21/2 = 5730 Years = 5730 Years

Carbon-14 RadiotracerCarbon-14 Radiotracer In pharmaceutical research carbon-14 is In pharmaceutical research carbon-14 is

used as a tracer to ensure that potential used as a tracer to ensure that potential drugs are metabolized without forming drugs are metabolized without forming harmful by-productsharmful by-products

The carbon-14 label should ideally form The carbon-14 label should ideally form part of the compound's molecular skeletonpart of the compound's molecular skeleton

Used in biological research, agriculture, Used in biological research, agriculture, pollution control and archeologypollution control and archeology

Classes of Carbon-14 Labelled Compounds

Carbon-14 labelled DrugsCarbon-14 labelled Drugs

Carbon-14 Building BlocksCarbon-14 Building Blocks

Carbon-14 Custom PreparationCarbon-14 Custom Preparation

LabellingLabelling Labelled building block (small molecule) then built up by Labelled building block (small molecule) then built up by

multi-stage organic synthesis on multi- mg scalemulti-stage organic synthesis on multi- mg scale

PurificationPurification Prep HPLC, Flash column chromatograhy, CrystallizationPrep HPLC, Flash column chromatograhy, Crystallization

AnalysisAnalysis HPLC, Radio-TLC, MS, NMRHPLC, Radio-TLC, MS, NMR

DispensingDispensing Solid, SolutionSolid, Solution

Radio-decompositionRadio-decomposition

PrinciplePrinciple

The compound itself and/or its immediate The compound itself and/or its immediate surroundings will absorb the radiation energy surroundings will absorb the radiation energy

Energy absorbed by the compound will excite Energy absorbed by the compound will excite the molecules which can break-up or react with the molecules which can break-up or react with other moleculesother molecules

The excited decomposition fragments may also The excited decomposition fragments may also react with other labelled compounds producing react with other labelled compounds producing other impuritiesother impurities

Energy absorbed by the immediate surrounding Energy absorbed by the immediate surrounding (often the solvent) can produce reactive species (often the solvent) can produce reactive species such as such as free radicalsfree radicals which can result in the which can result in the destruction of the labelled compounddestruction of the labelled compound

Mechanism of decompositionMechanism of decomposition

Fact:Fact: Compounds that are labelled with Compounds that are labelled with radioisotopes decompose faster than their radioisotopes decompose faster than their unlabelled counterpartsunlabelled counterparts

Consider: Half-life, Specific Activity (SA) Consider: Half-life, Specific Activity (SA) and equally shelf-life of the labelled and equally shelf-life of the labelled compoundcompound

Most important is shelf-lifeMost important is shelf-life-metabolic studies often require a -metabolic studies often require a

radiochemical purity of at least 98%radiochemical purity of at least 98%

Shelf-lifeShelf-life

The time during which a labelled The time during which a labelled compound may be used with compound may be used with confidence and safety, is important to confidence and safety, is important to both the user and the supplierboth the user and the supplier

The purity at which a radiolabelled The purity at which a radiolabelled compound ceases to be of use compound ceases to be of use depends on the applicationdepends on the application

The Specific Activity (SA) of The Specific Activity (SA) of CompoundsCompounds

Specific Activity (SA) - Specific Activity (SA) - the number of the number of radioactive transformations per unit mass radioactive transformations per unit mass of a compoundof a compound

In general, the higher the SA of a In general, the higher the SA of a compound, the greater the rate of compound, the greater the rate of decompositiondecomposition

This is constant for a given isotope This is constant for a given isotope in its in its atomic stateatomic state and cannot be altered by and cannot be altered by chemical or environmental changeschemical or environmental changes

Specific Activity (SA) of Specific Activity (SA) of moleculesmolecules

C

SA = 62.4 mCi/mmol

C C C CC C

[1-14C] compoundSA = 62.4 mCi/mmol

C C C CC C

[U-14C] compoundSA= 6 x 62.4 = 374.4 mCi/mmol

Decreasing the Specific Decreasing the Specific Activity (SA)Activity (SA)

C C C CC C

[1-14C] compoundSA = 62.4 mCi/mmol

C C C CC C

Adjusted SA = 62.4/6 = 10.4 mCi/mmol

Add 5 equivalents of inactive compound

Modes of Decomposition of Modes of Decomposition of Radiochemicals (1960)Radiochemicals (1960)

Primary (internal) decompositionPrimary (internal) decompositionnatural decaynatural decay

Results from the disintegration of the unstable Results from the disintegration of the unstable nucleus of the radioactive atomnucleus of the radioactive atom

Multiple carbon-14 label compounds on storage Multiple carbon-14 label compounds on storage produce minute decomposition productsproduce minute decomposition products

egeg 0.03% per year of [ 0.03% per year of [1414C]methylamine:C]methylamine:

1414CHCH331414CHCH33 → [ → [1414CHCH33

1414NHNH33] →] →1414CHCH33NHNH22

Yield of [1-Yield of [1-1414C]glycine from [2,3-C]glycine from [2,3-1414C]succinic acid C]succinic acid is very small!is very small!

Primary (external) decompositionPrimary (external) decomposition

The beta particles (ionizing radiation) The beta particles (ionizing radiation) interacts with molecules of labelled interacts with molecules of labelled compound surrounding the decaying nucleuscompound surrounding the decaying nucleus

Higher the SA , the greater the primary Higher the SA , the greater the primary decompositiondecomposition

Can add unlabelled (carrier) or other solvent Can add unlabelled (carrier) or other solvent to reduce SA or increase the number of non-to reduce SA or increase the number of non-labelled molecules near each labelled labelled molecules near each labelled moleculemolecule

Carbon-14 CompoundsCarbon-14 Compounds

Secondary decompositionSecondary decomposition

This type of decomposition is the most This type of decomposition is the most damaging mode and results from the interaction damaging mode and results from the interaction of labelled molecules - free radicals or other of labelled molecules - free radicals or other excited species produced by the radiationexcited species produced by the radiation

Difficult to control and is also the mode most Difficult to control and is also the mode most susceptible to minor variations of the susceptible to minor variations of the environmental conditionsenvironmental conditions

The low chemical weight of labelled compounds, The low chemical weight of labelled compounds, particularly at high SA , intensifies the problemsparticularly at high SA , intensifies the problems

Decomposition in aqueous Decomposition in aqueous solutionssolutions

Many radiochemicals are soluble only in Many radiochemicals are soluble only in hydroxylic solventshydroxylic solvents and water is the ideal and water is the ideal solvent!solvent!

These include: amino acids, carbohydrates and These include: amino acids, carbohydrates and nucleic acidsnucleic acids

Radiation chemistry of waterRadiation chemistry of water is therefore an is therefore an important consideration in the study of the important consideration in the study of the stability of radiochemicals in aqueous systemsstability of radiochemicals in aqueous systems

Since, the effect on the labelled compound of Since, the effect on the labelled compound of the reactive species produced is the prime the reactive species produced is the prime example of secondary decompositionexample of secondary decomposition

Ionizing radiation on waterIonizing radiation on water

Primary entities believed to result from the Primary entities believed to result from the radiolysis of water are:radiolysis of water are:

Hydronium ionsHydronium ions Hydrated electronsHydrated electrons Hydrogen atomsHydrogen atoms Hydroxyl radicalsHydroxyl radicals Hydroperoxy radicalsHydroperoxy radicals Molecular hydrogenMolecular hydrogen Hydrogen peroxideHydrogen peroxide

In solution ionization occurs along the In solution ionization occurs along the tracts of the beta particles in discrete tracts of the beta particles in discrete

pockets known aspockets known as SPURSSPURS

Ionizing radiation on waterIonizing radiation on water

Minimize decompositionMinimize decomposition

-reducing the number of solute-radical -reducing the number of solute-radical interactionsinteractions

-Achieved by lowering the temperature of -Achieved by lowering the temperature of storage and by the use of radical scavengersstorage and by the use of radical scavengers

-Free radical scavengers must react -Free radical scavengers must react preferentially and rapidly with the reactive preferentially and rapidly with the reactive species present in solution with no further species present in solution with no further reaction - reaction - must terminatemust terminate

Decomposition in organic Decomposition in organic solventssolvents

Decomposition in organic solventsDecomposition in organic solvents

The detailed mechanism of self-The detailed mechanism of self-decomposition of radiochemicals in decomposition of radiochemicals in organic solvents is unknown and is likely organic solvents is unknown and is likely to be to be complexcomplex

The transfer and absorption of the The transfer and absorption of the radiation energy is quite different and radiation energy is quite different and produces different forms of reactive produces different forms of reactive species from those in aqueous systemsspecies from those in aqueous systems

Organic Solvents Organic Solvents

Most widely used solvents include:Most widely used solvents include:

-Benzene (sometimes)-Benzene (sometimes)

-Toluene-Toluene

-Ethanol-Ethanol

-Methanol-Methanol

-Ethyl acetate-Ethyl acetate

-Pentane-Pentane

Decomposition in organic solventsDecomposition in organic solvents

Chemical purity of the solvent is critical (use high Chemical purity of the solvent is critical (use high purity solvents)purity solvents)

Trace of peroxide in the solution may cause total Trace of peroxide in the solution may cause total destruction of the labelled compound (avoid destruction of the labelled compound (avoid diethyl ether, or other ethers)diethyl ether, or other ethers)

Chlorinated hydrocarbons (chloroform) must be Chlorinated hydrocarbons (chloroform) must be avoided as solvents because of their potential avoided as solvents because of their potential quenching effectquenching effect

Chemical impurities may cause an increase in Chemical impurities may cause an increase in the rate of self-decomposition of the the rate of self-decomposition of the radiochemical on storageradiochemical on storage

Using BenzeneUsing Benzene

Benzene provides a positive effect on other Benzene provides a positive effect on other compoundscompounds

Many sensitive radiochemicals at high SA for Many sensitive radiochemicals at high SA for example steroids, fatty acids and hydrocarbons example steroids, fatty acids and hydrocarbons are remarkably stable towards self-radiolysis in are remarkably stable towards self-radiolysis in benzene solutionsbenzene solutions

The addition of a secondary solvent to benzene The addition of a secondary solvent to benzene solution of a radiochemical, normally added to solution of a radiochemical, normally added to increase the solubility of the compound in the increase the solubility of the compound in the solvent mixture, sometimes solvent mixture, sometimes leads to an leads to an acceleration of self-decompositionacceleration of self-decomposition

SteroidsSteroids

The addition of methanol to The addition of methanol to toluene/benzene solutions of labelled toluene/benzene solutions of labelled steroids has been observed to increase steroids has been observed to increase the rate of decomposition of these the rate of decomposition of these compoundscompounds

Possibly by increasing the lifetime of polar Possibly by increasing the lifetime of polar attacking attacking methoxy radicalsmethoxy radicals

Polar solvents such as methanol or water Polar solvents such as methanol or water greatly accelerate the rate of self-greatly accelerate the rate of self-decomposition of labelled steroidsdecomposition of labelled steroids

EstersEsters

Acidic compounds containing carboxyl Acidic compounds containing carboxyl groups may become esterified on prolonged groups may become esterified on prolonged storage in alcoholic solutionstorage in alcoholic solution

Formation of 24% [U-Formation of 24% [U-1414C]phenylalanine ethyl C]phenylalanine ethyl ester on storage of ester on storage of

[U-[U-1414C]phenylalanine in 95% ethanol at room C]phenylalanine in 95% ethanol at room temperature for 22 months temperature for 22 months

[[1414C]-Amino acids (AA)C]-Amino acids (AA) At low SA (<30 mCi/mmol) AA are best stored At low SA (<30 mCi/mmol) AA are best stored

as solids (freeze-dried) at 0as solids (freeze-dried) at 0°°C or lower C or lower temperaturestemperatures

Under nitrogen, argonUnder nitrogen, argon At higher SA best stored in aqueous solutions At higher SA best stored in aqueous solutions

containing 2% ethanol at a radioactive containing 2% ethanol at a radioactive concentration of 50-100 uCi/ml at +2concentration of 50-100 uCi/ml at +2°°C or frozen C or frozen at -20at -20°°C or lower temperatureC or lower temperature

If these conditions are followed the If these conditions are followed the decomposition rate of [decomposition rate of [1414C]-amino acids does not C]-amino acids does not exceed 2% per yearexceed 2% per year

Ethanol acts as a Ethanol acts as a radical scavengerradical scavenger

Molecular ClusteringMolecular Clustering

At +2At +2°°C in the unfrozen state the solute molecules C in the unfrozen state the solute molecules are free to move about in solutionare free to move about in solution

In solutions that are frozen slowly, say at -20In solutions that are frozen slowly, say at -20°°C to C to -40-40°°C, molecular clustering of the solute occurs C, molecular clustering of the solute occurs as freezing of pure solvent around the edge of the as freezing of pure solvent around the edge of the sample occurs firstsample occurs first

This clustering of the solute molecules is much This clustering of the solute molecules is much less marked in solutions that are frozen rapidly at less marked in solutions that are frozen rapidly at -196-196°°C (shelf-freezing)C (shelf-freezing)

Distribution of solute molecules (x) in aqueous Distribution of solute molecules (x) in aqueous and in frozen aqueous solutions – molecular and in frozen aqueous solutions – molecular

clustering effect on slow freezingclustering effect on slow freezing

Stability of [Stability of [1414C]-CompoundC]-Compound

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80%

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Effect of Specific ActivityEffect of Specific Activity

Decomposition of [14C]-Compound at 20°C100%

30%

90%

60%

20

55

120

Specific activities in mCi/mmol

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Effect of TemperatureEffect of Temperature

Stability of [14C]-Compound

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70%

90%

80%

-80º

-20º

20º

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Effect of Free Radical ScavengersEffect of Free Radical Scavengers

Decomposition of [14C]-Compound at 20ºC

100%

90%

80%

70%

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+ 3% ethanol

Aqueous solution

‘‘Ideal’ Radioactive Decomposition Ideal’ Radioactive Decomposition RatesRates

Screening Solvents: Screening Solvents: [14C]-Compound

0102030405060708090

RCP % (HPLC)

T=0 T=1 d T= 6 d T=12 d T=61 d

TIME

DCMTolueneACN/WaterTBMETHFTBMEEtOAc

Principal guides that will help minimize Principal guides that will help minimize decompositiondecomposition

Optimise storage conditions for chemical stability (correct pH, storage Optimise storage conditions for chemical stability (correct pH, storage under inert gas under inert gas etcetc))

As much as possible keep radiochemicals in the darkAs much as possible keep radiochemicals in the dark Store at low temperatures – solutions of radiochemicals should be stored Store at low temperatures – solutions of radiochemicals should be stored

cold but unfrozen (aqueous solutions at +2cold but unfrozen (aqueous solutions at +2°°C, ethanol solutions at C, ethanol solutions at -20-20°°C)C) Compounds of low chemical stability should be stored at -80Compounds of low chemical stability should be stored at -80°°C C Compounds in their natural physical state should normally be stored at Compounds in their natural physical state should normally be stored at -20-20°°CC Dilute the SA – a compound at high SA will decompose faster than at lower Dilute the SA – a compound at high SA will decompose faster than at lower

SASA Store as solutions – this effectively disperses the labelled molecules, Store as solutions – this effectively disperses the labelled molecules,

decreasing the effect of secondary decompositiondecreasing the effect of secondary decomposition Add radical scavengers or other stabilizers –when compatible with the use, Add radical scavengers or other stabilizers –when compatible with the use,

adding a radical scavenger (2-3% ethanol added to an aqueous solution) adding a radical scavenger (2-3% ethanol added to an aqueous solution) can lead to an increased shelf lifecan lead to an increased shelf life

Avoid reopening of vials, and warming/cooling cycle – if a radiochemical is 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 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 number of vials, keeping those to be used later in the refrigerator or freezer until requireduntil required