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Foreword
Amersham Biosciences has more than fifty years experience in the manufacture
and supply of radiochemicals. A continuing program of investigation into the
self-decomposition of radiochemicals on storage has ensured that these
compounds are supplied at the highest quality for research use. Much of the
information already obtained from these investigations has been made available to
users through publications and as part of the technical information supplied with
the labelled compounds. The aim of this review booklet, therefore, is to further
familiarize and remind the established user of radiochemicals of the problems of
self-decomposition. It is also intended for those investigators who are new to the
applications of radiochemicals. Their attention is drawn to the principles of
self-decomposition on storage, and to the methods, based both on theoretical
considerations as well as on experimental observations, which can be used to
control or minimize the problem.
By reading this review the user should be able to identify and establish the likely
optimum storage conditions for a particular labelled compound that is to be
used in a tracer experiment.
This booklet is an updated review based on the original publication by E.A. Evans,
Self-decomposition of radiochemicals (previously published by Amersham
Biosciences).
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Contents
Introduction
5
Radionuclides
1. The fraction of the energy absorbed
2. The specific activity of compounds
3. The exponential decrease in absorbed energy with time
Isotopes and their properties (related to those discussed in
this booklet) (Table)
6
6
6
6
6
Mechanisms of decomposition
Modes of decomposition of radiochemicals (Table)
7
8
Classification of decomposition
1. Primary (internal) decomposition
2. Primary (external) decomposition
3. Secondary decomposition
4. Chemical and microbiological decomposition
9
9
10
10
10
Decomposition of radiochemicals in solutions
1. Decomposition in aqueous solutions
2. Decomposition in organic solvents
11
11
13
Decomposition of radiochemicals in their natural physical state
14
Effects of temperature on decomposition
15
Rules to help minimize the decomposition of radiochemicals
1. Optimize storage conditions for chemical stability
2. Store at low temperatures
3. Dilute the specific activity
4. Store as solutions
5. Add radical scavengers or other stabilizers
6. Avoid reopening of vials and freeze/thaw cycles
Application of decomposition rules
17
17
17
17
18
18
18
18
Observations of self-decomposition
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Compounds labelled with Carbon-14
a. Amino acids
b. Carbohydrates and polyhydric alcohols
c. Nucleics
d. Long chain acids (fatty acids)
e. Steroids
f. Miscellaneous compounds
21
21
21
22
23
23
23
Compounds labelled with tritium
Storage of tritium-labelled compounds in their natural form
Storage of tritium-labelled compounds in solution
a. Amino acids
b. Carbohydrates
c. lnositol and inositol phosphates
d. Nucleosides
e. Nucleotide 5'-triphosphates
f. Vitamin D3 metabolites
g. Leukotrienes
h. Other tritiated compounds
i. Tritium exchange loss in solution
25
25
26
26
26
26
27
27
27
27
28
28
Compounds labelled with phosphorus-32, phosphorus-33, or sulfur-35
1. Phosphorus-32 labelled compounds
a. Formulation
Typical decomposition rates for the different formulations of
32P-labelled nucleotides (Table)
b. Temperature
c. Radioactive concentration
d. Specific activity
e. Nucleoside base
2. Phosphorus-33 labelled compounds
Radiochemical purity of [γ-33P]ATP over a 6-week period (Table)
3. Sulfur-35 labelled compounds
a. Amino acids
Percent radiochemical purity on storage of Redivue and standard formulations
of [35S]Methionine stabilized with 0.1% 2-mercaptoethanol (Table)
b. Thionucleotides
Effect of storage on radiochemical purity and incorporation in a multiprime
assay system of Redivue and standard formulations of [35S]dATPαS (Table)
Volatile impurities
c. Chemicals
28
28
29
30
31
31
31
32
32
33
33
33
35
35
36
36
36
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Compounds labelled with Iodine-125
a. Peptides and proteins
b. Thyroid hormones
Self decomposition of L-[3'-125I]Triiodothyronine and
L-[3'-5'-125I]Thyroxine in ethanol:water (3:1) at 20 °C (Table)
c. Steroids
Bolton and Hunter reagent for protein iodination
39
39
References
40
4
37
37
38
39
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Introduction
Radiolabelled compounds are widely used in biological, medical, and chemical
research. For such applications it is important that the radiochemicals used are of
appropriate purity and stability. Accepting that absolute purity is an ideal that
cannot be achieved in practice, the radiolabelled compound must have a purity
that is at least adequate for the experiment being conducted. From the viewpoint
of a radiochemical supplier, it is essential that compounds are of the highest
purity in terms of radiochemical and radionuclidic purity and as high as is
practicable in terms of chemical purity. Individual researchers should, likewise,
understand the purity of the radiochemical they are using so they can be confident
in its use. For all radiochemicals, the purity is normally highest at the time of final
purification. It is commonly reported on product specification sheets in terms of
percentage radiochemical purity as measured by chromatographic methods such
as thin-Iayer chromatography (TLC), high performance liquid chromatography
(HPLC), paper chromatography (PC) or gas-Iiquid chromatography (GLC). However,
on storage, decomposition reduces the purity, and it is important for researchers
to understand the rate and mechanisms of decomposition so that steps may be
taken to minimize it. If a researcher has any reason to doubt the radiochemical
purity of a labelled compound (for instance, its fitness for the intended use) it
should be re-analyzed before use. If this is impractical, advice from the supplier
should be sought. After storage it may be necessary to repurify a labelled
compound to meet the quality required, or to obtain a fresh supply.
The purpose of this booklet is to:
• Review the factors affecting the rate of decomposition of radiochemicals
labelled with tritium, carbon-14, phosphorus-32, phosphorus-33, sulfur-35,
and iodine-125.
• Describe some of the ways of minimizing the rates of decomposition.
• Provide examples of classes of compounds.
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Radionuclides
Decomposition depends in part on the amount of energy absorbed by the compound
during storage. There is, however, no direct correlation between the energy of the
emission and the rate of decomposition. This is because other factors are of
importance. These include:
1. The fraction of the energy absorbed
For the more energetic beta emitters such as phosphorus-32, much of the energy
escapes the system. On the other hand, almost complete total absorption of the
beta energy occurs with tritium compounds. It is not surprising therefore that
problems exist in the storage of tritium-labelled compounds. Gamma energy is, in
general, little absorbed by the compound itself or its immediate environment.
2. The specific activity of compounds
In general, the higher the specific activity of a compound, the greater the rate of
decomposition. This is particularly noticeable with tritiated compounds, where the
drive to higher specific activity for higher sensitivity has led to multiple labelling
(some tritium-labelled compounds containing up to eight tritium atoms per
molecule).
3. The exponential decrease in absorbed energy with time
As a radionuclide decays, so the emitted energy decays. This is only of significance
with short half-life isotopes such as phosphorus-32. It is not an important influence
in the longer-lived isotopes such as carbon-14 and tritium, and of little effect in
sulfur-35 or phosphorus-33.
Table 1 lists the nuclides commonly used in biological, medical, and chemical
research, and their important physical properties.
Table 1. Isotopes and their properties (related to those isotopes discussed in this booklet)
Property
14
3
125
Half-life
5730 years
12.3 years
C
H
32
33
35
59.9 days
14.3 days
25.6 days
87.5 days
I
P
P
S
Mode of decay
β
β
(EC)
β
β
β
Maximum β-energy
(MeV)
0.157
0.019
Electron
capture
1.71
0.249
0.168
Specific activity at 100%
isotopic abundance
2. 31 GBq/mA 1.07 TBq/mA
62.2 mCi/mA 28.9 Ci/mA
80.2T Bq/mA
2170 Ci/mA
336.2 TBq/mA 187.8 TBq/mA 54.95 TBq/mA
9086 Ci/mA
5075 Ci/mA
1485 Ci/mA
Common specific activities
for labelled compounds
37 MBq–3.7
GBq/mmol,
1–100
mCi/mmol
3.7 GBq–3.7
TBq/mmol,
0.1–100
Ci/mmol
3.7 GBq–74
TBq/mmol,
0.1–2000
Ci/mmol
74 GBq–222
TBq/mmol,
2–6000
Ci/mmol
55.5–92.5
TBq/mmol,
1500–2500
Ci/mmol
37 MBq–37
TBq/mmol,
1mCi–1000
Ci/mmol
Daughter nuclide (stable)
Nitrogen-14
Helium-3
Tellurium-125
Sulfur-32
Sulfur-33
Chlorine-35
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Mechanisms of decomposition
Compounds that are labelled with radioisotopes decompose faster than their
unlabelled counterparts. Although it is normal to select an isotope on criteria
including its half-life (or its inverse counterpart, the specific activity), an equally
useful concept is that of the shelf life (the time during which a labelled
compound may be used with confidence and safety) of the labelled compound.
It is the shelf life that is of importance, both to the user of the compound and to
the supplier of such compounds. The purity at which a radiolabelled compound
ceases to be of use depends a great deal on the application. Metabolic studies, for
instance, often require a radiochemical purity of at least 98%. Radioimmunoassay
can be successfully carried out with a radiochemical purity of 90% or less
(although it is preferable to have a radiochemical purity of at least 95%).
When comparing the decomposition of radiochemicals it is important to
consider the molar specific activity of compounds (e.g. MBq[mCi]/mmol rather
than MBq[mCi]/mg). The use of molar specific activity gives an appreciation of
the extent of labelling of a compound. For example, a tritium-labelled
compound of specific activity 1.073 TBq (29 Ci)/mmol has on average one
tritium atom per molecule. For a carbon-14 compound, a specific activity of
2.294 GBq (62 mCi)/mmol tells us that there is, on average, one carbon-14
atom per molecule. For a carbon-14 compound of specific activity 370 MBq
(10 mCi)/mmol, it may be a mixture of 10/62 of the molecules with one
carbon-14, and 52/62 of the molecules being unlabelled, or a smaller
proportion of multiply labelled molecules.
The modes by which radiolabelled compounds decompose and their
corresponding methods of control were classified in 1960 (1) (see Table 2).
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 surroundings (often the solvent)
can produce reactive species such as free radicals, which can then cause
destruction of the molecules of labelled compound. Whilst this is occurring,
chemical decomposition may also take place (radiolabelled compounds are
frequently supplied in solution, and not in their natural state. Hence although
an unlabelled compound may be stable for many years as a solid, its chemical
stability in solution may be far more limited).
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Table 2. Modes of decomposition of radiochemicals
Mode of decomposition
Cause
Method of control
Primary (internal)
Natural isotopic decay
None for a given specific activity
Primary (external)
Direct interaction of radioactive
emission (alpha, beta or gamma)
with molecules of the compound
Dispersal of labelled molecules
Secondary
Interaction of excited species
with molecules of the compound
Dispersal of labelled molecules;
cooling to low temperatures;
free radical scavenging
Chemical and
microbiological
Thermodynamic instability of
compound and poor choice of
environment
Cooling to low temperatures;
removal of harmful agents
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Classification of decomposition
1. Primary (internal) decomposition
Fig 1. Diagrammatical representation of radiochemicals at low and high specific activity,
and at high specific activity in a diluent.
This arises from the disintegration of the unstable nucleus of the radioactive
atom. For the radioisotopes being considered in this booklet, this method of
decomposition can normally be neglected, particularly for the longer half-life
isotopes carbon-14 and tritium. Even with multiple-labelled compounds, which
give rise to labelled impurities (one atom having decomposed, the other
labelled atom still being present) the build-up of impurities is still very slow.
For example, [1,2-14C]ethane produces only 0.03% [14C]methylamine per year
by isotope decay.
14
CH3-14CH3 è14CH3-NH2
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2. Primary (external) decomposition
This involves the direct interaction of the emitted particles (beta particles or
electrons for carbon-14, tritium, sulfur-35, phosphorus-32, and phosphorus-33)
with the molecules of labelled compound surrounding the decaying atom. If the
beta particles strike an unlabelled molecule and change it in some way, then only
a minor amount of unlabelled chemical impurity will be formed. This observation
tells us two things. Firstly, the higher the specific activity of a compound, the
greater the primary (external) decomposition becomes since the probability of the
beta particles striking a labelled compound is also increased. Secondly, it
provides a method of controlling the build-up of labelled impurities by
decreasing the probability of interaction of beta particles with labelled
compounds. For example, addition of unlabelled (carrier) compound to reduce
the specific activity or addition of a solvent to divert the energy of the beta
particles away from the labelled compound. However, correct choice of the
solvent or solvent mixture is vital to lessen the effect of secondary decomposition.
3. Secondary decomposition
This is commonly the greatest cause of the decomposition of radiochemicals,
and arises from the interaction of, for example, free radicals created by the
radiation, with labelled molecules. It is the most difficult mode of decomposition
to control and is also very easily influenced by small changes to the
environmental conditions. The low chemical weight of labelled compounds,
particularly at high specific activity, intensifies the problems.
4. Chemical and microbiological decomposition
It is often easy to concentrate on the effects of radioactivity on the decomposition
of radiochemicals, and to overlook the possibilities of chemical or microbiological
decomposition. This is particularly important with those compounds of known
instability (e.g. compounds easily oxidized should be protected under inert
gases). Also, those particularly sensitive to certain chemicals should be
protected from exposure to such compounds. In many cases, radiolabelled
compounds are stored as solutions at very low chemical concentrations (often
µg/ml or lower) and the need for high purity solvents is self-evident.
Microbial decomposition is commonly reduced by the sterilization of aqueous
solutions. Compounds susceptible to decomposition by light should be
protected by the use of opaque outer containers.
The combination of these processes can give rise to a range of decomposition
products, some of which were not present in the preparation of the compound.
For this reason, the repurification of a decomposed compound can be a problem,
particularly when a high proportion of the impurity has similar chromatographic
properties to the desired compound (since chromatographic methods are widely
used for the purification and repurification of radiolabelled compounds).
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Decomposition of radiochemicals in solutions
It has already been mentioned that many radiochemicals, particularly those at
high molar specific activity, are stored as solutions. This is partly due to their
slower decomposition in solution (greater dispersion) than in their natural form,
and partly due to the very small weight of such compounds, which makes their
handling in solution far more practical than handling the substances in their
natural form.
For example, a 3H-labelled steroid of molecular weight 360 and specific activity
1.11 TBq (30 Ci)/mmol) has a specific activity of 1.11 TBq (30 Ci)/360 mg,
which is 3.08 GBq (83.3 mCi)/mg.
Hence, a 37 MBq (1 mCi) vial of the compound contains only 12 µg of
compound. The practical difficulties of handling this very small weight are
formidable, but as a 37 MBq (1 mCi)/ml solution the material can be handled
with ease. Radiochemical decomposition would also be more rapid if it was
kept as the solid. Some products are specially formulated for direct use in
specific applications, for example, [1-14C]Chloramphenicol, CAT assay grade
(CFA754).
The use of solutions to store radiochemicals does not eliminate decomposition,
but does, in the main, lessen it. The rate of decomposition in solution is
governed by two main factors:
i. The rate of production of reactive species per mass of the labelled
compound, which depends on the specific activity of the compound.
ii. The rate of production of reactive species per ml of solution, which depends
on the radioactive concentration.
Both factors are important, but their overall effect depends very much on the
type of compound, the composition of the solution, the temperature, and the
general conditions of storage.
1. Decomposition in aqueous solutions
Many radiochemicals of interest to biochemists and biologists (such as amino
acids, carbohydrates, nucleosides, and nucleotides) are soluble only in
hydroxylic solvents of which water is the ideal solvent. Water is also a suitable
solvent as many of these compounds are used in aqueous media, so that little,
if any, reformulation of the radiochemical is required before its use.
Consequently, it is important to be aware of the radiation chemistry of water,
since the interaction of the reactive species formed with the labelled compound
is the classic example of secondary decomposition.
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Fig 2. Proximity of 'spurs' or 'pockets' of reactive species in solutions of radiochemicals
labelled with beta emitting radionuclides.
The action of ionizing radiation on water is well documented (2). Ionization
occurs along tracks of the beta particles (3) in discrete pockets known as spurs.
The weaker the energy of the radiation, the closer together the spurs become
(see Fig 2) and the greater the decomposition caused. The most damaging of the
reactive species believed to be formed is thought to be the hydroxyl radical (4).
This is evidenced by the hydroxylation of carbon-14 or tritium-labelled
phenylalanine, to produce tyrosine and dihydroxyphenylalanine (5).
In order to lessen the decomposition, it is necessary to lessen the number of
interactions between the damaging radicals and the molecules of the
radiochemical. This is normally achieved by lowering the temperature to +2 °C
(or as low as practicable without freezing the solution), diluting the radioactive
concentration (typical concentrations of 37 MBq (1 mCi)/ml are used), and by
adding radical scavengers. Ethanol is a common radical scavenger (typically as
a 2% solution in water) and has the advantage that it can be relatively easily
removed if a solely aqueous solution is required. The choice of a radical
scavenger is wide and considerable information is available on the interaction
rates of hydroxyl radicals and other reactive species produced by X- or gammaradiation (6–9). This data can be used as a guide for the selection of suitable
scavengers for reducing the self-decomposition of radiochemicals.
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2. Decomposition in organic solvents
The detailed mechanism of self-decomposition of radiochemicals in organic
solvents is not known, and is likely to be complex. The effect of radiation on
organic solvents is expected to be much different to that of aqueous systems
and would produce different forms of reactive species. The most widely used
solvents are ethanol, toluene, methanol, pentane, and ethyl acetate.
For many years benzene was a popular choice of solvent, providing good
storage characteristics for a wide range of radiochemicals. However, in the late
1970s concern over its toxic nature led to its replacement by toluene in cases
where there was no significant detrimental effect on the decomposition rate of
the compounds concerned.
The chemical purity of the solvent is critically important and freshly purified or
very high quality purchased solvents should be used. The presence of a
peroxide in the solution may cause total destruction of the radiochemical and
for this reason it is wise to avoid the use of diethyl ether, or other ethers, as
solvents. Chlorinated hydrocarbons such as chloroform are best avoided as
solvents because of their potential quenching effect.
Where a secondary solvent of higher polarity is needed to aid solubility of the
compound, an increase in decomposition rate is sometimes observed, possibly
because of the creation of longer-lived radicals. The decomposition rate of
labelled steroids increases on the addition of methanol to the benzene solution
(10). However, if ethanol is used as a radical scavenger, its addition is
beneficial to the radiochemical stability.
The compatibility of the radiolabelled compound with the organic solvent
must also be given careful consideration. For example, the storage of
[U-14C]phenylalanine in 95% ethanol at 20 °C for 22 months has been reported
to produce 24% of [U-14C]phenylalanine ethyl ester (11). Although strictly not
decomposition resulting from the presence of a radioactive isotope, the result is
a decrease in radiochemical purity, and is equally valid.
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Decomposition of radiochemicals in their
natural physical state
The rate of decomposition of a radiochemical in its natural physical state (solid,
liquid, or gas) depends on the chemical stability of the compound, its radiation
sensitivity, the energy of the emitted radiation, and the storage conditions. In
general, the weaker the energy of the radiation, the more it is absorbed and the
greater the damage caused. For a compound labelled with a β emitter at a
given specific activity under the same storage conditions in the natural state,
the rate of self-decomposition decreases in the following order:
Tritium > carbon-14 = sulfur-35 > phosphorus-33 > phosphorus-32
For those radiochemicals that can be freeze-dried (i.e. dispersed in air), the
radiochemical decomposition is usually lower than that of the crystalline solid,
in which individual molecules are closer together.
Some radiochemicals become colored on storage in their natural form.
Although the color is indicative of impurity, it is frequently present to a very
minor extent and does not affect the use of the compound. Examples include
the release of iodine from 14C- or 3H-labelled methyl iodide, the sometimes
straw-color of labelled benzene or acetic anhydride, and the purple color of
high specific activity sodium boro[3H]hydride (thought to be due to alteration of
the crystal lattice during its manufacture).
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Effects of temperature on decomposition
As a general rule, compounds, particularly organic compounds because of
their thermodynamic instability, are best kept at low temperatures. Chemical
reactions, such as radical-solute interactions, are normally decelerated by a
decrease in temperature as the activation energy for such reactions is raised;
hence, the chemical stability of a radiochemical will be increased by lowering
its temperature. It is normally beneficial to store radiochemicals in their natural
physical state or in solution at the lowest practicable temperature.
In the frozen state, at -40 °C, radical-solute interaction is substantially reduced.
However, another effect has to be considered when solutions, especially
aqueous solutions, of labelled compounds are frozen and then stored. This is
the effect of molecular clustering of the solute as solutions are frozen.
Figure 3 shows a diagrammatic feature of freezing solutions of compounds.
At +2 °C in the unfrozen state, the solute molecules are free to move about
the solution. In solutions that are frozen slowly, at say -20 to -40 °C, molecular
clustering of the solute occurs because 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 in liquid nitrogen (the
phenomenon of molecular clustering is readily demonstrated by slowly freezing
dilute solutions of a colored compound).
Fig 3. Distribution of solute molecules (x) in aqueous solutions and in frozen aqueous
solution – molecular clustering effect on slow-freezing; a. +2 °C, b. frozen quickly at -196 °C,
c. frozen slowly at -20 °C to -40 °C.
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The close proximity of the spurs (see page 12) in solutions of tritiated
compounds, means that molecular clustering has a very pronounced effect in
such solutions, compared with solutions of compounds labelled with higher
radiation energy emitting radionuclides.
Attempts to freeze tritiated compounds rapidly by immersion of their aqueous
solutions in liquid nitrogen followed by storage at -20 °C or -40 °C have not
been very successful. In many instances, decomposition is still observed to be
faster than in the unfrozen state at +2 °C, especially when a radical scavenger
is present in the solution (4).
Another effect to be avoided in frozen solutions is the separation of the solute
from the solvent. This can be caused either by the compound crystallizing in
the freezing process, or because a temperature gradient along the storage tube
causes the solvent to sublime to the cooler parts of the tube. This effect is
particularly noticeable in evacuated sealed ampoules and especially when
compounds are stored in organic solvents such as benzene.
An exception to the general rule that the lower the temperature the greater the
stability, is observed with the storage of certain radiochemicals that are
normally gases or vapors, which are prone to polymerization. Condensation of
such compounds by lowering the temperature can often accelerate their rate of
polymerization. This effect is most pronounced with compounds at a high molar
specific activity (e.g. [U-14C]Benzene and [3H]Methyl iodide).
The storage of radiochemicals in solution at very low temperatures (e.g. in
liquid nitrogen at -196 °C) has been used with good effect (14) but it is less
convenient than storage at +2 or -20 °C and is normally reserved for those
compounds of very low stability.
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Rules to help minimize the decomposition of
radiochemicals
To help users get the best possible use from their radiochemicals, we have
combined the principles discussed in previous sections of this booklet into six
principle rules that will help minimize the decomposition of radiochemicals.
1. Optimize storage conditions for chemical stability
Chemicals labelled with radioisotopes decompose by a combination of
chemical decomposition, affected by the chemical nature of the compound
and its environment, and by radiolytic decomposition caused by the
presence of the radioisotope. Storage conditions suitable for good chemical
stability should be used (e.g. correct pH, storage under inert gas). It must be
remembered that high specific activity means small chemical weight, and it
is often impractical to store such compounds in their normal physical state.
This results in many compounds being stored as solutions, and such storage
is more of a compromise than an optimization.
Radiochemicals should be kept in the dark (as much as is practical) and
protected from the adverse effects of any nearby chemicals. Solutions of
radiochemicals, particularly high specific activity ones, are especially
vulnerable to chemical decomposition in the presence of light and harmful
chemicals.
2. Store at low temperatures
Solutions of radiochemicals should be stored cold but unfrozen (e.g. aqueous
solutions at +2 °C, ethanol solutions at -20 °C). Compounds of a very low
chemical stability should be stored at -140 °C (the vapor above liquid
nitrogen). Compounds in their natural physical state should normally be
stored at -20 °C. If solutions are to be frozen, molecular clustering
(see page 15) is to be avoided.
3. Dilute the specific activity
Select a specific activity suitable for the planned use. In general, a
compound at high specific activity will decompose faster than at lower
specific activity.
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4. Store as solutions
Radiochemicals are often conveniently supplied and best stored as solutions.
This effectively disperses the labelled molecules, decreasing the frequency
of impact by excited species, thereby reducing secondary decomposition.
The correct choice of solvent or solvent mixtures is crucial in providing the
stabilizing effect.
5. Add radical scavengers or other stabilizers
When compatible with the use of the radiochemical, the addition of a radical
scavenger (e.g. 2–3% ethanol added to an aqueous solution) can lead to a
much longer shelf-life than is the case with water alone. Other stabilizers are
used as appropriate (e.g. antioxidants for those compounds susceptible to
oxidation, especially when in solution).
6. Avoid reopening of vials and freeze/thaw cycles
If a radiochemical is to be used over several weeks or months, it is advisable
to sub-aliquot it into a number of vials, keeping those to be used later in the
refrigerator or freezer until required.
Although not totally infallible, these rules provide a guide to the most effective
ways to minimize the decomposition of radiochemicals.
Application of decomposition rules
In practice, a combination of several or most of these rules can be used,
depending on the nature of the compound and its intended application.
Simplest to achieve is storage at low temperatures, particularly at + 2 or -20 °C.
Storage at -140 °C requires the use of liquid nitrogen flasks and is generally
less convenient than storage at +2 ° in refrigerators or -20 °C in freezers (some
use is made of storage at -80 °C, either short-term with dry ice, or in -80 °C
freezers). It is wise to store purchased radiochemicals at the temperature
recommended by the supplier. The supplier has taken into account the ease of
use of the radiochemical as well as its storage characteristics, and will have
stability data at the recommended temperature.
Protection from other chemicals and light is important, particularly for sensitive
compounds but also for all compounds supplied in solution. Radiochemicals at
high molar specific activity are present in a very dilute solution and can be
easily affected by exposure to reactive chemicals or by light. The more lightsensitive compounds are supplied in vials contained in an opaque outercontainer to lessen such decomposition.
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Alongside protection from light and other potentially harmful chemicals, the
optimization of chemical stability is a basic requirement. This is possible for
compounds in their natural physical state, but many compounds at high
specific activity are in solution, and as mentioned in the six rules (see page 17),
storage in solution is often a compromise.
The effects of radiation need to be considered and dispersal of the labelled
compound in a solvent is a good method to lessen radiation induced
decomposition. It also provides a method of handling compounds at high
specific activity. In general, conditions for good chemical stability should be
used, but with awareness that other factors, due to the effects of the radiation
present, must be taken into account.
Dilution of the specific activity is a way of dispersing the molecules of labelled
compound by the addition and thorough mixing of the unlabelled compound.
For many applications, however, it is not appropriate to do this (e.g. RIA and
receptor-binding studies require high specific activity tracers). It is advisable to
purchase sufficient labelled compound for the study and use it within the shelf
life of the compound (normally indicated on batch analysis sheets) than to buy
a larger quantity but suffer the problems caused by storage past its shelf life.
When it is feasible to dilute the molar specific activity by addition of the
unlabelled compound, the weight of unlabelled (carrier) compound to be
added can be calculated from the following equation:
W = Ma 11
A
where
1
A
W=
M=
a =
A=
weight of carrier to be added (mg)
molecular weight of the compound
total amount of radioactivity in the sample (mCi)
molar specific activity of the compound at the higher specific
activity (mCi/mmol)
A1 = molar specific activity required for the diluted compound
(mCi/mmol)
Practicalities of the mixing depend on the formulation of the compound. Liquids,
gases, and vapors can be added directly to their labelled counterparts and will
mix evenly themselves. Labelled compounds in solution can be mixed with the
unlabelled compound, either by direct addition or by addition in the same
solvent as the labelled compound. The dilution of the specific activity of solids is
conveniently carried out by dissolving the labelled compound in a suitable
solvent, addition of the carrier compound, swirling to ensure complete
dissolution, and removal of the solvent by rotary evaporation or by freeze-drying.
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Free radical scavengers and other stabilizers provide an important improvement
in extending shelf life. Radical scavengers must react rapidly and preferentially
with the reactive species present in solution. The most common scavenger is
ethanol, which is effective at concentrations as low as 1–3% in aqueous or
organic solvents (4,12). Ethanol has the advantage that it is easily removed by
evaporating or freeze-drying the solution.
Other free-radical scavengers have been tested. These include benzyl alcohoI
(4,11,15), sodium formate (4,11), glycerol (14), cysteamine (14), ascorbic acid
(4), and mercaptoethanoI (11,12). For many applications, the use of a labelled
compound in water:ethanol (98:2) is not a problem because the ethanol
percentage in the actual experiment is very low (often < 0.1 %). But for some
applications researchers require the labelled compound in water without
ethanol. These are cases where ethanol may be detrimental to the study.
For this reason, other stabilizers have been developed for use with radiolabelled
compounds. These include polymer beads PT6-271*, which is used to provide
good stability on storage of [3H]Inositol, and pyridine-3,4-dicarboxylic acid,
which is used with good effect in stabilizing 35S-labelled amino acids
(e.g. L-[35S]Cysteine).
The practice of removing vials of radiolabelled compounds from cold storage,
particularly -20 °C, allowing them to warm to room temperature before sampling,
and returning them to their cold storage, can, over several such cycles, lead to
a lowering of radiochemical purity. This can be caused by the freeze/thaw
cycles themselves, exposure to air and light, or accidental contamination with
adverse chemicals or biochemicals. If multi-sampling over several days is to be
done, it is advisable to sub-aliquot the total activity into several vials, or to
purchase several smaller vials from the supplier. The vials that are not
immediately required should be stored as recommended by the supplier.
*PT6-271 is a solid stabilizer developed by Amersham Biosciences for use with myo-[3H]Inositol.
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Observations of self-decomposition
This section of the booklet deals with the self-decomposition of compounds
labelled with carbon-14, tritium, sulfur-35, phosphorus-33, phosphorus-32,
and iodine-125. The aim of the section is to show how longer shelf-lives can be
achieved by wise application of the methods to control self-decomposition, and
show the value of the six rules described earlier (see page 17). Within each
isotope a selection of examples will be used to illustrate these points.
Compounds labelled with carbon-14
Compounds labelled with carbon-14 are widely used in tracer studies,
particularly in the area of metabolism where high radiochemical purity and
good stability on storage are very important. The relatively low molar specific
activity of carbon-14 compounds (compared with tritium) reduces the selfdecomposition problem. However, this is offset by the higher decay energy of
carbon-14 and the possibility of near 100% isotopic abundance in many
compounds. Observations of the self-decomposition of carbon-14 compounds
have led to optimization of storage conditions for various classes of labelled
organic compounds and are discussed below.
a. Amino acids
Amino acids labelled with carbon-14 are best stored in aqueous solution
containing 2% ethanol at a radioactive concentration of 1.85–3.7 GBq
(50–100 mCi)/ml at +2 °C. Such storage conditions normally provide a
decomposition rate not exceeding 2% per annum (15).
Storage of amino acids in dilute hydrochloric acid can protect against bacterial
growth but can result in greater rates of self-decomposition. Hence a
compromise in storage conditions is needed for optimum overall stability.
If there is a requirement to use only a sample of the activity, it is advisable to
subaliquot the remaining material as required and to maintain these
subaliquots under optimum storage conditions.
b. Carbohydrates and polyhydric alcohols
When stored in 3% aqueous ethanol solution at -20 °C, 14C-labelled
carbohydrates and polyhydric alcohols show normally less than 2%
decomposition per annum (15).
In a number of cases, no detectable decomposition is seen after storage
for over 1 year. For example, D-[U-14C]Glucose at 11.8 GBq (320 mCi)/mmol
in water with 3% ethanol at -20 °C for 17 months showed no measurable
decomposition (15).
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c. Nucleics
Purine and pyrimidine bases labelled with carbon-14 are among the most
stable of labelled organic compounds. For example [U-14C]Adenine at
> 7.4 GBq (200 mCi)/mmol stored in 2% aqueous ethanol at +2 °C shows
a decomposition rate far below 1% in 1 year.
Nucleosides labelled with carbon-14 are fairly stable when stored in solution at
+2 °C or -20 °C. The position of label, as well as storage conditions, may have
an influence on the overall stability.
Nucleotides labelled with carbon-14, such as nucleotide 5'-monophosphates
and particularly the 5'-triphosphates, are chemically unstable and great care is
needed in choosing the best conditions for their storage and analysis. Storage
at -20 °C is most advisable for these products.
A major decomposition route for ribonucleosides and deoxy ribonucleosides is
by splitting the base from the sugar in acidic conditions. This was the subject of
a comprehensive review by Capan (16).
In general, 14C-labelled nucleotides are best stored in sterilized aqueous
solutions containing 2% ethanol at -20 °C, at ~ pH 7 and at 1.85–3.7 MBq
(50–100 mCi)/ml. The decomposition rate for these products is usually less
than 1% per month.
As expected, the stability is lower for compounds at very high specific activity
(18.5–22 GBq, 500–600 mCi/mmol) than at lower specific activities, other
conditions of storage being the same.
Nucleotide sugars labelled with carbon-14 are even more sensitive to chemical
decomposition than the 14C-labelled nucleoside triphosphates. Nucleoside
diphosphate sugars are easily hydrolyzed by mild acid treatment. Uridine
diphosphoglucose (UDPG), whose behavior is typical of this class of compound,
is completely hydrolyzed to UDP and D-glucose in 0.01 M HCI at 100 °C for
10 min. Nucleoside diphosphate sugars that possess a free hydroxyl group at
carbon 2 of the sugar group are also alkali labile (17) (e.g. hydrolysis of UDPG
to glucose-l,2-cyclic phosphate and UMP is rapid in concentrated ammonia
even at 0 °C).
When reactions involving nucleoside diphosphate sugars are followed by
chromatographic analysis, it is often convenient to spot samples at intervals
onto chromatography paper and subsequently develop the chromatogram
overnight. Experiments have shown that the decomposition of these
compounds is accelerated on paper, especially under mildly alkaline
conditions.
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Nucleoside diphosphate amino-sugars are much more resistant to alkaline
hydrolysis because there is no free hydroxyl group at carbon 2 in the amino
sugar. For example, the initial rate of hydrolysis of UDP-N-acetyl glucosamine
in 0.1 M NaOH at 60 °C is about 0.7% per hour, whereas for UDPG in
0.1 M NaOH, decomposition is virtually instantaneous at room temperature.
The optimum conditions for storage of 14C-labelled nucleoside diphosphate
amino-sugars are in sterilized aqueous solutions containing 2% ethanol at
-20 °C.
Bivalent metal ions such as Cu2+ also cause significant increases in the rate of
hydrolysis of nucleoside diphosphate sugars.
d. Long chain acids (fatty acids)
The most stable formulations for [14C]Arachidonic acid are in toluene solution
containing an anti-oxidant, or in ethanol solution. The product is also supplied
in toluene solution without anti-oxidant, but is not as stable. In all cases the
product should be stored under nitrogen in the absence of light.
e. Steroids
The recommended storage conditions for 14C-labelled steroids are in toluene
solution at -20 °C. When toluene solution alone gives insufficient protection,
ethanol is added.
f. Miscellaneous compounds
Catecholamines, drugs, vitamins, aliphatic, and aromatic compounds are among
the miscellaneous 14C-labelled compounds. By following carefully the general
methods recommended for the control of self-decomposition, most 14C-labelled
compounds can be stored for long periods (12 months and more) with less than
2% decomposition. Compounds that are rather sensitive to decomposition in
solution are often best stored as freeze-dried solids. However,
[14C]Benzylpenicillin, an especially sensitive compound, showed 8%
decomposition during 6 months as a freeze-dried solid at -20 °C. Even lower
storage temperatures (-140 °C and below) are recommended in such cases.
S-Adenosyl-L[carboxyl-14C]Methionine is very sensitive to both chemical and
radiochemical conditions. The product is most stable in dilute sulfuric acid at
pH 2.5–3 at low temperature.
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At 1.85 GBq (50 mCi)/mmol and 370 kBq (10 mCi)/ml, the decomposition
rates versus temperature are as follows:
at +25 °C
decomposition rate 21% per week
at +2 °C
decomposition rate 2% per week
at -20 °C
decomposition rate under 0.1% per week.
The data clearly shows the importance of not leaving solutions of sensitive
compounds at room temperature for longer than is required. The problem can
be overcome by cooling the vial with ice during the use of the compound, or by
subaliquoting and keeping the subaliquots under optimum storage conditions.
Other general observations regarding the decomposition of 14C-labelled
compounds are as follows:
i. In the solid or liquid form it is frequently possible to obtain improved stability
by storing a carbon-14 compound in a thin film. Dispersion increases
stability and protects against self-decomposition.
ii. Radiation catalyzes the formation of free radicals and decomposition by free
radical reactions can sometimes proceed at great speed.
iii. The polymerization of labelled aliphatic iodides such as [14C]methyl iodide at
high specific activity (2.2 GBq, 60 mCi/mmol) is not observed, in contrast to
tritiated methyl iodide, which at specific activities above 111 GBq (3 Ci)/mmol
may spontaneously form a yellow solid. However, a gradual darkening of the
color occurs due to the release of free iodine, together with the formation of
[14C]methane and [14C]ethane as gaseous impurities.
iv. Labelled aldehydes are usually sensitive to oxidation and are often better
stored as derivatives.
v. Barium [14C]carbonate and sodium [14C]carbonate are two raw materials from
which many organic compounds labelled with carbon-14 are derived. Such
compounds are not normally regarded as unstable in their radioactive form.
However, decomposition can occur by loss of the isotope, usually as [14C]carbon
dioxide, which is exchanged with the carbon dioxide in the atmosphere.
Studies by Barakat and Farag (18) have shown that barium [14C]carbonate,
if stored as a solid, should be kept under vacuum in the presence of sodium
hydroxide pellets. In contrast to normal storage recommendations for labelled
organic compounds in the solid state, care should always be taken to
decrease the exposed surface area of the stored [14C]carbonate. It is best to
store sodium [14C]carbonate as an aqueous solution in sealed ampoules. It
does not then exhibit loss of radioactivity by exchange on storage.
In summary, the observed self-decomposition of most organic compounds
labelled with carbon-14 is usually quite small provided the recommended
precautions are taken to control the decomposition rate.
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Compounds labelled with tritium
The high absorption of tritium beta radiation energy and the ability to achieve
specific activities in the range of 1.11–3.14 TBq (30–85 Ci)/mmol and up to
7.4 TBq (200 Ci)/mmol, makes the control of self-decomposition of tritiumlabelled compounds a more difficult task than for compounds labelled with
carbon-14.
A number of generalizations can be made regarding the self-decomposition on
storage of tritium-labelled compounds.
1. All tritiated compounds at high specific activities (with a few specific exceptions)
should be stored and handled in solution.
2. Tritiated compounds at high specific activity stored in solution are very
sensitive to decomposition by chemical and biochemical degradation. This is
due to their extremely low chemical concentration.
3. Self-decomposition can be accelerated by the effect of molecular clustering
(see page 15) on storage of frozen solutions.
4. Radical scavengers markedly improve the storage characteristics of tritiated
compounds in solution.
5. Some tritiated compounds are prone to gradual loss of the label as tritiated
water due to exchange with the aqueous solution, but procedures such as
freeze-drying can overcome this problem. Further details on exchange
losses of tritium-labelled compounds are given on page 28.
Storage of tritium-labelled compounds in their natural form
The great majority of tritium-labelled compounds are stored as solutions
because of their improved stability in this medium. The few exceptions tend to
be labelling intermediates such as [3H]Acetic anhydride, [3H]Methyl iodide, and
Sodium boro[3H]hydride. These products are stored in their natural form for a
combination of reasons but largely because of greater chemical stability and
ease of use (no reformulation) for labelling work. Their use is almost entirely as
labelling intermediates, so they do not come into the category of end products
to be used directly for investigative work. Satisfactory stability is achieved, even
with Sodium boro[3H]hydride, at specific activities over 1.85 TBq (50 Ci)/mmol
and storage at room temperature.
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Storage of tritium-labelled compounds in solution
It is essential that most tritium-labelled compounds, particularly those at high
molar specific activity, are stored as solutions to provide acceptable protection
against self-decomposition. Additional factors that affect stability include the
storage temperature (see page 15), the presence of free-radical scavengers
(see page 18), molecular clustering (see page 15), solvent purity, the
compound's chemical stability, and pH. The following classes of tritium-labelled
compounds and decomposition rates help to illustrate many of these points.
a. Amino acids
Tritiated amino acids at low specific activity (< 18.5 GBq, 500 mCi/mmol)
can be stored as freeze-dried solids at +2 °C or -20 °C, with less than 5%
decomposition per annum. At higher specific activity, storage is recommended at
+2 °C as a sterilized aqueous solution in water containing 2% ethanol, and at a
concentration of 37 MBq (1 mCi)/ml.
The general rules are as follows:
• The higher the specific activity, the greater the rate of decomposition for that
compound.
• The addition of 2% ethanol has a very beneficial effect, due to its radical
scavenging property.
• The higher the radioactive concentration, the higher the rate of
decomposition.
b. Carbohydrates
The majority of tritiated carbohydrates, at 37 MBq (1 mCi)/ml, have a
decomposition rate of between about 0.05 and 1% per month. Those with
the highest decomposition rate (between about 1 and 3% per month) all lack
ethanol, with a consequently greater effect of free radicals on these compounds.
An alternative formulation (containing ethanol) is provided for those applications
where ethanol is tolerated, and its beneficial effect is clear (e.g. L-[6-3H]Glucose
decomposition rate improves from about 1% per month in water only, to about
0.1% with the addition of 3% ethanol).
c. Inositol and inositol phosphates
The most noticeable difference in the decomposition rates is the much higher
rate (about 2.5% per month) for myo-[2-3H]inositol in sterilized water compared
with rates well below 1% per month in the presence of 10% ethanol or the
polymer PT6-271: even at specific activities one magnitude higher.
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Researchers use a choice of formulations for myo-[2-3H]Inositol. For some
applications ethanol is not tolerated, and an aqueous solution is the usual
choice. More generally labelled myo-[3H]Inositol, at much higher specific
activity (2.96–4.44 TBq, [80–120 Ci]/mmol) needs stabilization either by
10% ethanol or by PT6-271. The decomposition rates are very acceptable,
and virtually identical in these two formulations. There is no comparative figure
for myo-[3H]Inositol at 2.96–4.44 TBq (80–120 Ci)/mmol in water only, but the
decomposition rate is expected to be far in excess of 2–5% per month.
The variables for inositol phosphates are compound structure, specific activity,
and position of label. All are stored at +2 °C in water:ethanol 9:1 at
37 MBq (1 mCi)/ml. Most have decomposition rates between about 0.5
and 1% per month – quite acceptable for tritium-labelled compounds.
The effect of higher specific activity is to raise the decomposition rate.
d. Nucleosides
There are a number of variables including storage temperature, formulation,
specific activity, position of label, and compound. Consequently, a number of
comparisons and contrasts can be made, but the general points to aid longer
shelf-life re-emerge. In general, 50%, 10%, or 2% ethanol provides improved
stability. Also, for most items the higher the specific activity, the higher the
decomposition rate.
e. Nucleotide 5'-triphosphates
In general, compounds with a high proportion of tritium at the 8 position of the
purine ring have a higher decomposition rate due to greater release of the label
by exchange with the solvent. Also, higher specific activity tends to give rise to
higher decomposition rates (8-labelled purine compounds excepted), but care
must be exercised when comparing chemically different compounds.
f. Vitamin D3 metabolites
The variation in decomposition rates for this group of products is largely in line
with specific activity, but individual compound chemistry may also play some
part. Optimization of the formulation of these chemically sensitive
radiochemicals greatly reduces their decomposition rates.
g. Leukotrienes
Decomposition rates tend to follow specific activities, which exceed
3.7 TBq (100 Ci)/mmol for most of these compounds. Readers can make
their own comparisons, bearing in mind possible differences caused by the
chemistry of the compounds.
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h. Other tritiated compounds
Tritium-labelled steroids at 37 MBq (1 mCi)/ml in ethanol or toluene/ethanol
mixtures at -20 °C frequently do not exceed 1% decomposition per month.
Chemically unstable compounds, such as vitamin A or noradrenaline, require
storage at -20 °C in dark containers.
i. Tritium exchange loss in solution
There is wide variation in the generation of tritiated water from different groups
of compounds, the differences being due to the location of the label and the
chemistry of individual compounds.
This variation was illustrated in Section e. by the higher decomposition rates of
nucleoside 5'-triphosphates labelled in the 8 position of the purine ring.
Compounds labelled with phosphorus-32, phosphorus-33,
or sulfur-35
Compounds labelled with relatively short half-life radionuclides are less likely to
be stored for long periods before use than compounds labelled with the longer
lived radionuclides carbon-14 and tritium. Impurities that may be present in
compounds labelled with phosphorus-32, phosphorus-33, or sulfur-35 are
therefore more often likely to have arisen during the preparation of these
compounds. Although the half-life of the three radionuclides is short, the
primary (internal) effect of decay in producing impurities is minimal. This is
because multiple labelling is rare.
1. Phosphorus-32 labelled compounds
Compounds labelled with phosphorus-32 are normally used within a few weeks
of their preparation because of the short half-life (14.3 days) of the radionuclide.
Historically, compounds were best stored in the form supplied at -20 °C or below.
If the total contents of the vial are not intended for use in one experiment then
the solution should be aliquoted upon receipt and each aliquot stored at -20 °C
until required. This procedure avoids repeated freeze/thaw cycles that increases
the rate of decomposition in standard formulation compounds.
Nucleotides labelled with phosphorus-32 comprise the largest group of
compounds labelled with phosphorus-32 commercially available. These
compounds are essential tools used in the development of various techniques
now employed for studies in molecular biology (19). The stability of 32P-labelled
nucleotides is affected by a number of factors as follows:
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a. Formulation
There are three main formulations for 32P-labelled nucleotides.
Standard formulation
For many years 32P-labelled nucleotides were supplied in water:ethanol (1:1) at
74 MBq (2 mCi)/ml. In this formulation, decomposition at -20 °C is normally
less than 2% per week. However, with developments in techniques, it was
found increasingly necessary to remove the aqueous ethanol and reconstitute
the nucleotide at a higher radioactive concentration in reaction buffer. This
process can easily lead to substantial chemical decomposition.
Stabilized formulation
In order to prevent this, and to supply a more convenient product, Amersham
Biosciences introduced 32P-labelled nucleotides in stabilized aqueous solution at
a radioactive concentration of 370 MBq (10 mCi)/ml. This formulation was
developed to allow use of the product direct from the vial. In addition, the
stabilizing component, 2-mercaptoethanol, is used in many biological systems
and does not inhibit experimental reactions. Decomposition rates of 32P-labelled
nucleotides, even at specific activities greater than 185 TBq (5000 Ci)/mmol, are
typically not in excess of 2% per week at -20 °C.
Nucleotides labelled with phosphorus-32 are often sold as stabilized aqueous
solutions, shipped in dry ice for storage by the customer at -20 °C. However, it is
of significant advantage both to the shipper and to the customer if radiolabelled
nucleotides are supplied at ambient temperature and stored in an unfrozen form.
Redivue formulation
In 1993, Amersham Biosciences developed and introduced the RedivueTM
range for convenience and stability. Redivue 32P-labelled nucleotides are
supplied as aqueous solutions incorporating both a highly visible red dye and a
stabilizer, enabling storage at +2 °C. The freeze-thaw cycle and the need to
subaliquot were therefore eliminated.
Redivue products are supplied at 370 MBq (10mCi)/ml. The additional feature
of Redivue is that products can be shipped ambient rather than frozen on dry
ice, so they are ready for use on receipt.
The colored solution of the Redivue formulation makes the solution more easily
visible during manipulation. However, the dye used must not interfere with any
process in which the radiochemical might be used.
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The stabilizer concentration is sufficient to reduce radiolytic decomposition of
the radiolabelled organic compound, while not being so high as to materially
interfere with the reaction systems where the radiochemical is to be used.
Preferred concentrations in liquid compositions are in the range of 1 mM to 1 M,
particularly 10 to 100 mM. Used in these concentrations, the preferred
compounds have proved effective stabilizers—particularly for nucleotides.
The stabilizer helps to prevent the dye from fading, and the dye improves the
visibility of the radiochemical. The dye and the stabilizer may act synergistically
to improve the stability of the radiolabelled organic compound.
Redivue and standard aqueous solution containing 5 mM 2-mercaptoethanol
[α-32P]dCTP at 110 TBq (3000 Ci)/mmol, 370 MBq (10 mCi)/ml were tested for
radiochemical purity and incorporation in a random-prime DNA labelling
reaction. The Redivue formulation was stored at +4 °C and the standard
formulation was stored at -20 °C.
The tests were carried out at weekly intervals from the first day of sale and the
results are shown in Table 3.
Table 3. Typical decomposition rates for the different formulations of
P-labelled nucleotides
32
Percent radiochemical purity
Redivue
Standard
MultiprimeTM DNA labelling
(% incorporation)
Redivue
Standard
Day 7
86
84
72
74
Day 15
87
67
63
67
Day 22
81
75
68
63
Due to the handling and convenience benefits, the Redivue formulation now
accounts for the majority of 32P-labelled nucleotides supplied by Amersham
Biosciences.
Regardless of formulation, observations have shown that pH is a crucial factor
in the stability of 32P-labelled nucleotides, possibly because of the extremely low
chemical concentration of most products. Below pH 7.5, decomposition can
exceed 1% per day—even at -20 °C.
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b. Temperature
Temperature can have a very marked effect on the stability of 32P-labelled
nucleotides. For standard formulations that are stored at -20 °C or below,
decomposition rates are normally less than 2% per week. At room temperature
(~ 20 °C) the rate of decomposition can be as high as 20% per week, but the
variation that is observed clearly indicates that other factors beside temperature
are influencing the rate. It is possible that impurities, even at a low level, may
have a catalytic effect on decomposition. However, 32P-labelled nucleotides are
sufficiently stable, even at 37 °C, to allow reactions in biological systems to take
place without the formation of sufficient decomposition products to present
problems in the interpretation of the experimental data. The formulation of the
reaction system is a major factor in the observed stability.
Redivue 32P-labelled nucleotides are far more tolerant of temperature
fluctuations. Samples of [α-32P]dTTP (AA0007) were stored at -20 °C, +4 °C
and room temperature for three weeks. The samples stored frozen at -20 °C
and in the fridge at +2 °C showed < 10% decomposition, whereas the room
temperature samples had decomposed ~ 20%.
c. Radioactive concentration
The majority of 32P-labelled nucleotides are supplied in solution at
370 MBq (10 mCi)/ml. Amersham Biosciences also supplies some products
at 740 MBq (20 mCi)/ml and 1.48 GBq (40mCi)/ml, specifically formulated for
particular techniques. At these higher concentrations, decomposition should
not exceed 3% per week in the period preceding the reference date if stored
at -20 °C. Beyond this, however, the rate of decomposition would be expected
to accelerate in comparison with more dilute formulations.
d. Specific activity
Variations in stability due to specific activity are observed only in extreme
cases. Nucleotides labelled with phosphorus-32 at low specific activity
(1.85 TBq [50 Ci]/mmol) show a greater long-term stability than products at
above 37 TBq (1000 Ci)/mmol. However, within the recommended usable life
of the product, very little difference is seen, although specific activity may affect
the nature of the decomposition products formed. At high specific activity,
these decomposition products are probably formed as a result of radiolytic
effects. Even under adverse storage conditions, when substantial breakdown is
observed, the chemical amounts are such that the products cannot be
identified. The behavior in chromatographic systems of impurities observed in
many high specific activity nucleotides suggest that they arise by the
radiolytically induced modification of the base moiety. At lower specific
activities, hydrolysis of the phosphate chain is apparent and orthophosphate,
nucleoside mono- and di-phosphates can be observed.
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e. Nucleoside base
The base present does affect stability, although guanosine and deoxyguanosine
triphosphates are less stable than the other nucleotides. In general the
ribonucleotides are more stable than the deoxyribonucleotides but the
difference in stability only becomes apparent at temperatures above 0 °C.
In general, 32P-labelled nucleotides, if maintained under recommended
conditions, have a degree of stability that allows their effective use in many
biological applications, and decomposition products do not appear to have any
inhibitory effect on the rate and efficiency of the reaction of the remaining
nucleotide. However, probably because of a combination of the comparatively
high energy of the beta particles and the high specific activity, and therefore
very low chemical concentration, they are more susceptible to variations from
ideal storage conditions than are compounds labelled with many other isotopes.
This susceptibility gives rise to a greatly accelerated rate of decomposition due
to a cumulative effect of several of the factors discussed above.
2. Phosphorus-33 labelled compounds
The nature of compounds labelled with phosphorus-33 is very similar to that of
compounds labelled with phosphorus-32 because the actual molecule and
position of the label are the same. However, the specific activities are
significantly lower.
The factors that affect the stability of 33P-labelled nucleotides are formulation,
storage temperature, specific activity, and radioactive concentration. These
factors have already been discussed. Please see the individual sections under
Phosphorus-32 labelled compounds for more information.
Nucleotides labelled with phosphorus-33 are available as a stabilized aqueous
solution containing 5 mM 2-mercaptoethanol, and also as Redivue formulations,
which are described in the phosphorus-32 section (see page 28).
Due to the lower specific activity of the product and the lower beta energy,
products labelled with phosphorus-33 tend to have lower rates of decomposition
and longer shelf-lives than their phosphorus-32 counterparts. They also benefit
from a longer half-life (25.6 days).
Table 4 shows the radiochemical purity of [γ-33P]ATP over a six-week period.
The Redivue formulation was stored at +2 °C and the standard formulation was
stored at -20 °C.
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Table 4. Radiochemical purity of [γ-33P]ATP over a 6-week period
Time (weeks)
Radiochemical purity (%)
of [γγ-33P]ATP (AH9968)
Redivue formulation
stored at +2 °C
Radiochemical purity (%)
of [γγ-33P]ATP (BF1000)
standard formulation
stored at -20 °C
0
95
98
2
94
89
4
92
88
The data shows that the decomposition rate of the Redivue formulation is far
slower than the standard formulation, even at a higher storage temperature.
3. Sulfur-35 labelled compounds
The major 35S-labelled compounds of interest are high specific activity
(37 TBq [1000 Ci]/mmol) amino acids and, to a lesser extent, thionucleotides.
A small range of 35S-labelled chemicals is also available.
a. amino acids
Interest in investigating various aspects of protein synthesis has led to the
widespread use of L-[35S]Methionine and L-[35S]Cysteine at very high
(> 37 TBq [1000 Ci]/mmol) specific activities.
These products are produced biosynthetically from sulfur-35 and a micro-organism,
followed by harvesting, hydrolysis, and purification. Both [35S]Methionine and
[35S]Cysteine are extremely susceptible to chemical degradation, accelerated by
radiolytically generated free radicals. Such degradation can be minimized by a
suitable combination of storage temperature and added stabilizer.
As a general rule, the lower the storage temperature achievable the slower
will be the rate of decomposition. L-[35S]Methionine stabilized with
2-mercaptoethanol at 555 MBq (15 mCi)/ml will decompose at ~ 2% per
week at -80 °C and will show < 1% decomposition per week at -140 °C,
while at -20°C the rate of decomposition may well exceed 10% per week.
For L-[35S]Cysteine stabilized by potassium acetate and DTT , the rates of
decomposition would be ~ 6% per week for storage at -80 °C, which reduces to
1% per week when stored at -140 °C. This is more than double the rates of
decomposition compared to those of an equivalent L-[35S]Methionine, stabilized
with potassium acetate and mercaptoethanol.
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A number of chemicals have been used to inhibit the degradation effect of free
radicals on 35S-labelled amino acids. For some years 2-mercaptoethanol was
the additive of choice for methionine and dithiothreitol the additive of choice for
cysteine. These compounds are still used in some formulations and have an
acceptable stabilizing effect. However, investigations at Amersham Biosciences
have shown that a greatly enhanced stability is attained by the use of pyridine3,4-dicarboxylic acid as a stabilizing agent. [35S]Methionine stabilized with
this reagent at 15 mM concentration and with 2-mercaptoethanol at 0.1%
decomposes at a rate of < 2% per month at -80 °C and 1% per week at -20 °C.
For [35S]Cysteine, the rate of decomposition is retarded by the addition of the
same reagent along with 5 mM dithiothreitol and 20 mM potassium acetate
to ~ 2% per week at -80 °C.
The major impurity observed on the decomposition of [35S]Methionine is
[35S]Methionine sulfoxide. [35S]Methionine also gives rise to small amounts of
an as yet unidentified impurity. In certain applications this impurity has an
effect disproportionate to its concentration. In some protein translation systems
it has been shown to incorporate to produce labelled proteins that cause
artifactual bands on autoradiograms of electrophoresis gels of the translation
products. These bands appear at ~ 47 kD and may mask the presence of
genuine bands of interest. In [35S]Methionine stabilized by 2-mercaptoethanol
alone this impurity will typically appear at a rate of up to 1% per week at
-80 °C; the addition of pyridine-3,4-dicarboxylic acid reduces the rate of
formation of this impurity to negligible proportions.
In the mid-1990s Amersham Biosciences extended the Redivue range to cover
[35S]Methionine. These products have the additional dye and stabilizer that
enables the product to be stored at +2 °C. Typical decomposition rates are 1%
per week for +2 °C and 1% per month for -80 °C storage.
Samples of Redivue [35S]Methionine and standard [35S]Methionine stabilized
with 0.1% 2-mercaptoethanol were stored at +4 °C and -20 °C. The samples
were tested at regular intervals for % radiochemical purity and the results are
shown in Table 5.
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Table 5. Percent radiochemical purity on storage of Redivue and standard
formulations of [35S]Methionine stabilized with 0.1% 2-mercaptoethanol
Percent radiochemical purity
[35S]Methionine
After 7 days
After 14 days
After 25 days
Redivue at +4 °C
93
90
84
84
Standard at +4 °C
52
10
3
-
Standard at -20 °C
84
63
41
27
After 32 days
Amersham Biosciences also developed Pro-mix L-[35S]in vitro Cell Labelling Mix,
which is a mixture of purified [35S]Methionine and [35S]Cysteine (70:30). Two
formulations are available, Redivue and stabilized aqueous solution. Typical
rates of decomposition for the stabilized aqueous solution stored at -80 °C are
3% per month for [35S]Methionine and 2% per week for [35S]Cysteine. These
figures reduce to 1% per month for [35S]Methionine and 1% per week for
[35S]Cysteine for the Redivue formulation when stored at +4 °C.
b. Thionucleotides
Thio analogs of nucleotides labelled with sulfur-35 to high specific activity
(> 37 TBq [1000 Ci]/mmol) in either the alpha or gamma positions on the
phosphate chain are still used for some applications in molecular biology,
particularly manual sequencing and in situ hybridization. They can be
substituted for 32P-labelled nucleotides in many DNA and RNA labelling and
sequencing systems. In cases where the highest degree of sensitivity
phosphorus-32 gives is not essential, there are advantages to be gained in
using 35S-labelled nucleotides. Inherent in the isotope are the longer half-life
(87.4 days) and the lower beta energy, which both reduces risks in handling
and leads to much higher resolution in autoradiography with little sacrifice of
time. Additionally, 35S-labelled nucleotides decompose at a much slower rate
than their phosphorus-32 equivalents. At 370 MBq (10 mCi)/ml 35S-labelled
nucleotides in 20 mM dithiothreitol solution will typically decompose at
< 2% per month when stored at -80 °C. At -20 °C the rate of decomposition
should not exceed 1% per week. However, 35S-labelled nucleotides at high
radioactive concentration (1.48 GBq [40 mCi]/ml) are best stored at -140 °C,
where 20 mM dithiothreitol solutions will degrade at no more than 1% per week;
at -80 °C decomposition of such solutions should not exceed 4% per week.
A limited range of Redivue α-35S-labelled dATP and α-35S-labelled dCTP
products are available. These have the benefit of the Redivue dye and
stabilizer, which enables them to be stored at +2 °C.
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Samples of [35S]dATPαS were stored at +40 °C for 24 h, then at room
temperature for 24 h, followed by long term storage at +2 °C. Samples
were formulated as Redivue and as a standard aqueous solution containing
20 mM DTT. Both samples were tested for RCP and incorporation in a
multiprime assay system over a period of 14 weeks. The results shown in
Table 6 illustrate the enhanced stability of the Redivue product.
Table 6. Effect of storage on radiochemical purity and incorporation in a
multiprime assay system of Redivue and standard formulations of [35S]dATPαS
αS
[35S]dATPα
Week 2
Week 4
Week 8
Week 14
Redivue
Standard Redivue
Standard Redivue
Standard Redivue
Standard
% radiochemical purity
92
92
88
58
85
40
85
30
% incorporation in
multiprime assay
56
53
71
56
71
39
72
38
Volatile impurities
Both amino acids and nucleotides labelled to high specific activity with sulfur-35
are known to give rise to small amounts of volatile radioactive impurities on
storage. Quantities of these impurities are typically < 0.1% of the total activity
even after several weeks storage; workers should nevertheless be aware of this
and take suitable precautions (e.g. open vials and handle in ventilated
enclosures, wear gloves, etc).
c. Chemicals
Chemicals labelled with sulfur-35 are supplied as low specific activity solids
(< 3.7 GBq [< 100 mCi]/mmol). Typical decomposition rates for these
35
S-labelled products when stored desiccated at room temperature are
approximately 1–2% during the half-life of the radionuclide (87.5 d).
For example, the radiochemical purity of [35S]Heparin (SJ193) was measured
by paper electrophoresis over a 16-week period. The initial radiochemical purity
was 96% and this had dropped to 92% after 16 weeks storage at room
temperature in a desiccator.
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Compounds labelled with iodine-125
lodine-125 decays by electron capture to stable tellurium-125, with a half-life of
59.9 d. The process involves the transfer of an orbital electron to the nucleus
of the atom with the emission of energy (0.03 MeV) in the form of weak X-rays.
Compounds labelled with Iodine-125 can be stored for acceptable lengths of
time, although the rates of radiochemical decomposition are usually greater
than the rate of nuclear decay. Labelling with iodine-125 usually involves the
introduction of a ‘foreign’ iodine atom to the compound of interest (important
exceptions to this include the thyroid hormone thyroxine). The introduction of
an iodine atom normally involves either an oxidative reaction or use of an
125
I-labelled conjugation reagent.
It is normally necessary to remove by-products formed in these processes
to enhance radiochemical stability and product performance. Since the
carbon-iodine bond is considerably weaker than the carbon-hydrogen bond,
the major product of decay, particularly with direct iodination, is usually iodine
itself. The characteristics of the molecule being labelled have a large impact
on its decomposition and the ways this decomposition affects the use of the
labelled material.
The two major areas in which Iodine-125 is used are (a) peptide and protein
labelling, and (b) thyroid hormones.
a. Peptides and proteins
Small peptides (up to 30 amino acids) can usually be labelled at one specific
amino acid residue with one atom of iodine. By use of suitable purification
techniques (normally chromatographic) impurities can be removed to yield a
carrier-free 125I-labelled compound with good radiolytic and chemical purity.
The best test for an 125I-labelled compound will always be the functional test to
which it will be applied.
For larger peptides and proteins, such as growth factors, specific labelIing is
often not possible. However, it is important to control the amount of iodine-125
incorporation to avoid excessive radiolytic decomposition and undue distortion
of the tertiary structure of the protein. This is an important factor in retaining
immunoreactivity or receptor-binding properties, features that can suffer from
multiple iodination. The stability and successful use of a protein labelled with
iodine-125 depends on the conditions used for its labelling and purification, as
well as its formulation and the subsequent careful handling and storage of the
product.
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Important aspects to consider include the following:
• Preservation of disulfide bonds when present (avoid reducing agents).
• Selection of suitable solvents (affecting the folding pattern of proteins).
• Control of proteolytic damage (by the presence of protease inhibitors).
• Minimizing decomposition by avoiding freeze-thaw cycles.
• Prevention of losses caused by the protein or peptide sticking to glass
(use silanized glass vessels or plastic vials).
• Use of the best storage form (solution or freeze-dried) and storage temperature.
• Storage at correct pH (which affects the protein or peptide, and can affect its
stability).
The overall stability of an iodine-125 labelled peptide or protein depends on all
these factors. The particular nature of the peptide or protein, and any special
requirements, dictate which has the greatest influence.
b. Thyroid hormones
Detailed studies have been carried out (20) on the stability of the
radioiodine-labelled iodothyronines. Thus, [3'-1251]triodothyronine (T3) at
55.5 GBq (1500 mCi)/mg stored in aqueous solution containing 75% ethanol
at 11.1–14.8 GBq (300–400 mCi)/ml showed about 2% decomposition during
one week at 20 °C or at -20 °C. Only 1% iodide was observed to form during
4 weeks, the major impurity being identified as 3,3'-[3'-125I]diiodothyronine
(reversed T2). [125I]Thyroxine (T4) at 2.96 GBq (80 mCi)/mg stored under
similar conditions showed ~ 5% decomposition after 4 weeks at 20 °C.
Again, only 1% iodide was observed, together with T3 and reversed T2 as major
identified products. These and other results are shown in Table 7 (21).
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Table 7. Self decomposition of L-[3'-125I]Triiodothyronine and
L-[3'-5'-125I]Thyroxine in ethanol:water (3:1) at 20 °C
Compound
Specific activity,
GBq/mg (mCi/mg)
Radioactive
concentration,
GBq/ml (mCi/ml)
Storage
time,
weeks
% decomposition
Iodide
Other
L-[3'-125I]Triiodothyronine
55.5 (1500)
11.1–14.8 (0.3–0.4)
1
2
4
8
1
2
2
5
8–10
12–15*
L-[3'-5'-125I]Thyroxine
2.96 (80)
11.1–14.8 (0.3–0.4)
1
2
4
8
1
2
1–2
1–2
3–5
5–7†
L-[3'-5'-125I]Thyroxine
14.8 (400)
14.8 (0.4)
2
4
1
1–2
7†
* Main decomposition products include 3,3'-[3'-125I]diiodothyronine (reversed T2) and two other equal
% iodine-125 impurities of unidentified structure.
† Main impurities include [125I]T3, [125I] reversed T2, and one unknown.
c. Steroids
Conjugates of steroids labelled with iodine-125 are used in radioimmunoassay
(e.g. (O-carboxymethyl)oximino-(2-[125I]iodohistamine), derivatives of testosterone,
cortisol, estradiol, and the 11a-hemisuccinate-(2- [125I]iodohistamine) and
11α-glucuronide-[125I]tyramine derivatives of progesterone).
Solutions of these compounds at approximately 74 TBq (2000 Ci)/mmol and
3.7 MBq (100 µCi)/ml in methanol:water (9:1) undergo < 5% decomposition in
4 weeks when stored at 2 to 4 °C.
Bolton and Hunter reagent for protein iodination (20,21)
Bolton and Hunter reagent is a solution of N-succinimidyl-3-(4-hydroxy-5[125I]iodophenyl) propionate in very dry benzene at a specific activity of
approximately 74 TBq (2000 Ci)/mmol and 185 MBq (5 mCi)/ml. It undergoes
about 10% decomposition in 2 weeks when stored at 5 to 10 °C. At lower
temperatures, in frozen benzene, the decomposition is accelerated (15–20% in
2 weeks) due to the labelled reagent being unprotected by the benzene in the
frozen state.
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References
1. Bayly, R.J. and Weigel, H., Self-decomposition of compounds labelled with radioactive
isotopes. Nature, 188, 384–387 (1960).
2. Thomas, ].K., Elementary processes and reactions in the radiolysis of water. Advances
in Radiation Chemistry, 1, 103–198 (1969).
3. Collison, E. and Swallow , A.J., The action of ionizing radiations on organic compounds.
Quarterly Reviews of the Chemical Society, 9, 311–327 (1955).
4. Evans, E.A., Tritium and its Compounds. 2nd edition, Butterworths, London,
pp. 642–782 (1974).
5. Waldeck, B., [3H]Dopa in [3H]tyrosine with high specific activity. Journal of Pharmacy
and Pharmacology, 23,.64–65 (1971).
6. Swallow, A.J., Radiation Chemistry of Organic Compounds, Pergamon Press, Oxford,
p. 380 (1960).
7. Bolt, R.O. and Carroll, J.R., Radiation Effects on Organic Material, Academic Press,
New York, p. 576 (1973).
8. Ebert, M., et al. Pulse radiolysis, Academic Press, London, p. 319 (1965).
9. Anbar, M. and Net A, P.,A compilation of specific bimolecular rate constants for the
reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals with inorganic and
organic compounds in aqueous solution, International Journal of Applied Radiations and
Isotopes, 18, 493–523 (1967).
10. Geller, L.E. and Silberman, N., Some factors involved in the decomposition of labelled
steroids on storage, Journal of Labelled Compounds, 5, 66–71 (1969).
11. Bayly, R.]. and Evans, E.A., Storage and stability of compounds labelled with
radioisotopes, Amersham Biosciences Review No.7, (1968).
12. Evans, E.A. and Stanford, F.G., Stability of thymidine labelled with tritium or carbon-14,
Nature, 199, 762–765 (1963).
13. Sheppard, G., Sheppard, H.C. and Stivala, ].F., The stability of thymidine, uridine
and their related nucleotides labelled with tritium, Journal of Labelled Compounds, 10,
557–567 (1974).
14. Apelgot, S. and Frilley, M., Heterogeneity of frozen thymidine solutions, Journal de
Chimie Physique, 62, 838–844 (1965).
15. Sheppard, G., The self-decomposition of radioactively labelled compounds, Atomic
Energy Review, 10, 3–66 (1972).
16. Capon, B., Mechanism in carbohydrate chemistry, Chemical Reviews, 69, 407–498
(1969).
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17. Paladini, A.C. and Leloir, L.F., Uridine-diphosphate-glucose, Biochemical Journal, 51,
426–430 (1952).
18. Barakat, M.F. and Farag, A.N., In-laboratory production of some carbon-14-labelled
compounds, Atomic Energy Establishment, Cairo, (Report No. AREAEE-156) p. 37 (1972).
19. Sambrook, J., Fritsch, E.F., and Maniatis, T., Methods in Molecular Cloning, Cold
Spring Harbor Laboratory Press, (1989).
20. Stanford, F.G., Stability of compounds labelled with iodine-125 or -131, The
Radiochemical Centre Limited, (TRC Report No.354). p. 18 (1974).
21. Bolton, A.E. et al. Three different radioiodination methods for human spleen ferritin
compared, Clin. Chem., 25, 1826–1830 (1979).
41

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