Working document QAS/07.242
September 2007
RESTRICTED
Draft general International Pharmacopoeia monograph for
Radiopharmaceuticals
Revision
(September 2007)
DRAFT FOR COMMENT
Please send any comments on the revision of this draft general monograph for
Radiopharmaceuticals to Dr S. Kopp with a copy to Ms M.-L. Rabouhans, Quality
Assurance and Safety: Medicines, Medicines Policy and Standards, World Health
Organization, 1211 Geneva 27, Switzerland; fax: (+41 22) 791 4730 or e-mail:
[email protected] and [email protected] by 10 November 2007.
© World Health Organization 2007
All rights reserved.
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The draft may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in part or
in whole, in any form or by any means outside these individuals and organizations (including the organizations’
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Please send any request for permission to:
Dr Sabine Kopp, Quality Assurance & Safety: Medicines (QSM), Department of Medicines Policy and Standards
(PSM), World Health Organization, CH-1211 Geneva 27, Switzerland.
Fax: (41-22) 791 4730; e-mail: [email protected].
The designations employed and the presentation of the material in this draft do not imply the expression of any opinion
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shall not be liable for any damages incurred as a result of its use.
Working document QAS/07.242
page 2
SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/07.242
Draft general International Pharmacopoeia monograph for Radiopharmaceuticals
Revision
Date
Draft revision of general monograph
mailed out for comments
September-October 2007
Collation of any comments received
October 2007
Presentation to WHO Expert Committee on October 2007
Specifications for Pharmaceutical
Preparations
Further follow-up action as required
October 2007 - …
Working document QAS/07.242
page 3
RADIOPHARMACEUTICALS
Introduction
The World Health Organization and the International Atomic Energy Agency (IAEA)
have been working jointly on specifications for Radiopharmaceuticals. Following
consultation and discussion, it was agreed that this work should include inter alia revision
of the general monograph in The International Pharmacopoeia and the preparation of
monographs for individual radiopharmaceuticals. Meanwhile, for the main volumes of
the Fourth edition of The International Pharmacopoeia, published in December 2006, the
section on Monographs for Radiopharmaceuticals consists of the general monograph for
Radiopharmaceuticals as included in the 3rd edition.
A draft revised general monograph for Radiopharmaceuticals has now been prepared by
the IAEA together with a first set of individual draft monographs for about 30
radiopharmaceutical preparations (more drafts are in preparation) for addition to the 4th
edition of The International Pharmacopoeia. These texts are now being circulated by
WHO for comment in line with the usual consultative process for monograph
development. As noted within the documents however, the WHO Secretariat has not, as
yet, adapted these texts to the format and style of The International Pharmacopoeia. This
will be carried out at a later stage. Comments are therefore invited on the technical
content of the draft monograph texts.
In addition to publishing these monographs in the section on Radiopharmaceuticals in a
future Supplement to The International Pharmacopoeia, it is intended that they also form
part of a joint IAEA/WHO publication that would also include other texts relevant to the
manufacture and use of radiopharmaceuticals. In including the monographs in such a
"stand-alone" publication, it would be necessary to supplement them with relevant
supporting texts from The International Pharmacopoeia. These would include, for
example, the General Notices, the general monographs for Parenteral Preparations and
Capsules, selected Methods of Analysis (such as 1.13 Determination of pH, 1.14.4 High
performance liquid chromatography, 3.4 Test for bacterial endotoxins).
Working document QAS/07.242
page 4
Draft general monograph for:
Radiopharmaceuticals
[Note from the Secretariat: Before inclusion in The International Pharmacopoeia this
draft general monograph will be adapted to the usual pharmacopoeial format, layout and
editorial style. An updated table of the physical characteristics of radionuclides will be
included.]
This general monograph is intended to be read in conjunction with the individual
monographs on radiopharmaceutical preparations. A radiopharmaceutical preparation that
is subject of an individual monograph in The International Pharmacopoeia complies with
the general requirements stated below.
Radiopharmaceuticals are unique medicinal formulations containing radioisotope which
are used in major clinical areas such as oncology, myocardial perfusion and infections.
The facilities and procedures for the production, use, and storage of radiopharmaceuticals
are generally subject to licensing by national and/or regional authorities. This licensing
will generally include regulations for the pharmaceutical preparations and for the
radioactive materials. Additional regulations may apply for issues such as transportation
or dispensing of radiopharmaceuticals. Each producer or user must be thoroughly
cognizant of the national requirements pertaining to the articles concerned. GMP
guidelines are available in Quality assurance of pharmaceuticals, Volume 2: Good
manufacturing practices and inspection (WHO, Geneva, 2004). In addition refer to IAEA
publications on safe handling and production of radioisotopes.
Radiopharmaceuticals are radioactive and can pose a risk to the personnel who prepare
and administer them and the patients to whom they are administered. Specialized
techniques are required to minimize the risks to personnel. All personnel involved in any
part of the operation are required to have appropriate additional training specific. The
maintenance personnel and support staff such as the cleaner should receive specific
instruction and appropriate supervision whilst in the operational areas. Risk to patient
should be minimised. It is essential to ensure that reproducible and clinically reliable
results will be obtained. All operations should be carried out or supervised by personnel
who have received expert training in handling radioactive materials.
Definition
Radiopharmaceuticals can be divided into four categories:
Radiopharmaceutical preparation A radiopharmaceutical preparation is a medicinal
product in a ready to use form suitable for human use which contains a radionuclide. The
radionuclide is integral to the medicinal application of the preparation, making it
appropriate for one or more diagnostic or therapeutic applications.
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Radionuclide generator A system in which a daughter radionuclide (short half-life) is
eluted (separated) from parent radionuclide/s (long half-life) and later used for
preparation of radiopharmaceutical for medicinal purpose.
Radiopharmaceutical precursor Any radionuclide produced for the radiolabelling
process with a resultant product aimed for medicinal use.
Kit for radiopharmaceutical preparation In general a vial containing essential predispensed precursor/s, in general pre-sterilized, pre-validated products to which the
appropriate radionuclide is added and diluted before medical use. In most cases this is a
multidose vial which may require additional steps including boiling or filtration. The kits
are designed for immediate use after preparation.
See Annex for terminology applied to radiopharmaceuticals.
Dosage form
See The International Pharmacopoeia 4th edition (Ph. Int.) for more details on various
dosage forms. In general radiopharmaceuticals are parenteral preparations however there
are RP which are oral solutions or capsules and other forms. Therefore they should
conform to the details outlined in these sections of the Ph. Int. Certain specific points
related to RP are highlighted here.
Parenteral RP solutions are sterile, pyrogen-free liquids solutions, or suspensions forms
containing one or more radioisotope, packaged in a suitable container and stored in
suitably shielded outer container. These are in either single-dose or multidose containers.
It should be noted that although RP emit radiation they themselves are not self-sterilizing
and, therefore, require the same consideration as normal parenteral preparation.
Most RP are intended for ‘immediate use’. All the technetium based RP and positron
emission tomography RP (PET) are prepared and used within 12 hours. This presents
different set of challenges as it is not possible to comply with sterility test before the
product is released for patients use. These parenterals therefore should be prepared under
strict aseptic condition under well validated systems. It is recommended that sterility
testing should continue to be performed however retrospectively. The main focus would
be operator competency, strict aseptic practices and relevant parametric release criteria.
Many of these are beyond the scope of the Ph. Int. and reference should be made to other
IAEA publications.
RP Labelling
The following information should appear on the label of the immediate container for
example, vial or syringe (p-primary packaging and s-secondary packaging):
•
•
•
•
“Caution –Radioactive –Material.” - p and s
The name of the radiopharmaceutical preparation - p and s;
The route of administration- p and s;
The statement that the product is radioactive- p and s;
Working document QAS/07.242
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•
•
•
•
•
•
•
The total radioactivity present at a stated dated and time- p and s;
The expiry date or expiry period- p and s;
A batch or lot number- p and s;
In case of solutions, the total volume together with dosage form- p and s;
Special storage requirements with respect to temperature and light- s;
In some cases name and concentration of any added microbial preservatives- s;
Any additional national or regional legal labelling requirements- p and s.
NOTE: In the case of a solution, instead of a statement of the total radioactivity, a
statement of the radioactive concentration (for example, in MBq per ml of the solution)
may be given.
The shipment of radioactive substances is subject to special national and international
regulations as regards to their packaging and outer labelling. See IAEA publication for
further details.
Radiation Shielding
Adequate shielding must be used to protect laboratory personnel from ionizing radiation.
Instruments must be suitably shielded from background radiation.
Alpha and beta radiations are readily shielded because of their limited range of
penetration, although the production of Bremsstrahlung by the latter must be taken into
account. The range of alpha and beta particles varies inherently with their kinetic energy.
The alpha particles are mono-energetic and have a range of a few centimetres in air. The
absorption of beta particles, owing to their continuous energy spectrum and scattering,
follows an approximately exponential function. The range of beta particles in air varies
from centimetres to metres.
The secondary radiation produced by beta radiation upon absorption by shielding
materials is known as Bremsstrahlung and resembles soft X-rays in its property of
penetration. The higher the atomic number or density of the absorbing material, the
greater the energy of the Bremsstrahlung produced. Elements of low atomic number
produce low-energy Bremsstrahlung, which is readily absorbed; therefore, materials of
low atomic number or of low density, such as aluminium, glass, or transparent plastic
materials, are used to shield sources of beta radiation.
Attenuation of gamma radiation in matter is exponential and is expressed in terms of halfvalue layers. The half-value layer is the thickness of shielding material necessary to
decrease the intensity of radiation to half its initial value. A shield of 7 half-value layers
is of a thickness that will reduce the intensity of radiation to less than 1% of its
unshielded intensity of activity. Gamma radiation is commonly shielded with material of
high atomic number such as lead and tungsten.
The intensity of gamma radiation is reduced according to the inverse square of the
intervening distance between the source and the point of reference. Radioactive materials
of several gigaBecquerel (GBq) strength can be handled safely in the laboratory by using
Working document QAS/07.242
page 7
proper shielding and/or by arranging for the maximum practicable distance between the
source and the operator by means of remote-handling devices.
Storage
Radiopharmaceuticals should be kept in well-closed containers and stored in an area
assigned for the purpose. The storage conditions should be such that the maximum
radiation dose rate to which persons may be exposed is reduced to an acceptable level.
Care should be taken to comply with national regulations for protection against ionizing
radiation.
1. Radiopharmaceuticals should be kept in well-closed containers and stored in an
area assigned for the purpose.
2. The radiopharmaceutical preparation should be stored in a glass vial, ampoule or
syringe that is sufficiently transparent to permit the visual inspection of the
contents.
3. It should be clearly indicated if the storage is at room temperature (defined by Ph.
Int. below 30°C), refrigerated (2-8°C), and in few cases kept frozen.
4. The container should be shielded with appropriate lead container to comply with
ALARA principle.
5. The storage conditions should be such that the maximum radiation dose rate to
which persons may be exposed is reduced to an acceptable level.
6. Care should be taken to comply with national and international regulations for
protection against ionizing radiation.
7. Glass containers may darken under the effect of radiation.
Expiry Date
The special nature of a radiopharmaceutical requires that it must be assigned an expiry
period (or an expiry date), beyond which its continued use is not permitted. The expiry
period so designated is fixed on the date of manufacture. The expiry period depends on
the radiochemical stability and the content of longer-lived radionuclide impurity in the
preparation under consideration. At the end of the expiry period, the radioactivity will
have decreased to the extent where insufficient radioactivity remains to serve the
intended purpose or where the dose of active ingredient must be increased so much that
undesirable physiological responses occur. In addition, chemical or radiation
decomposition may have reduced the radiochemical purity to an unacceptable extent.
Also the radionuclide impurity content may be such that an unacceptable radiation dose
would be delivered to the patient. The use of products beyond their expiry periods is
therefore inadvisable.
Manufacture
In general ways of manufacturing radionuclides for use in RP are:
Nuclear fission Nuclides with high atomic number are fissionable and the common
reaction is the fission of uranium-235 by neutrons in a nuclear reactor e.g.iodine-131,
molybdenum-99 and xenon-133. Radionuclides from such a process must be carefully
controlled in order to minimize the radionuclidic impurities.
Working document QAS/07.242
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Charged particles bombardment of target materials is increasingly common in
cyclotrons or in accelerators. The composition and purity of the target material will
determine the relative percentages of the principal radionuclide and ultimately the
radionuclidic purity. For very short lived radionuclides including the PET tracers the
determination of the chemical state and purity of radionuclide before patient use is
difficult. Therefore before use of these radionuclides in clinics, extensive validations and
strict operational conditions are essential. Strict control of range of specified quantity and
quality is also essential. Any subsequent change in operational conditions should be revalidated.
Neutron or charged particle bombardment of target materials in nuclear reactors and
particle accelerators (cyclotrons). The desired nuclear reaction will be influenced by the
energy of the incident particle the isotopic composition and the purity of the target
material.
Radionuclide generator systems provide availability of short-halved life clinically
useful radionuclide by separation of the daughter radionuclide from a long-lived parent
by chemical or physical separation.
Sterilization and radiopharmaceuticals
The general Ph. Int. principles apply to radiopharmaceuticals. However, for
radiopharmaceuticals (RP) containing a radionuclide of very short life (especially
positron emission tomography tracers [PET]) these would pose considerable constraints if
general requirements stated in Ph. Int. When possible, terminal sterilization is
recommended for RP. The main concern would be the practical safety of handling high
levels of radiation during the terminal sterilization process.
Further considers are fundamental when dealing with thermolabile, biological or
autologous radiolabelled products. In most of these cases strict aseptic process and
appropriate level of validation would be essential to insure safety of final
radiopharmaceutical for patient use. This is beyond the scope of this document and
specific advice on this can be found in other IAEA publications.
In radiopharmaceutical practices there is a wide use of filtration method, however caution
is advised. Establishing the integrity testing of final filtration units using Bubble point
technique is fundamental. However the process of test is destructive and cause spillage
and spread of radioactive materials which is also unacceptable. The levels of all risks
must be carefully considered. Expert advice and due care on these topics is of paramount
importance.
Addition of bacteriostatic agents
injections of radiopharmaceuticals are commonly supplied in containers that are sealed to
permit the withdrawal of successive doses on different occasions. The Ph. Int. normally
requires that such injections should contain a suitable bacteriostatic agent in a suitable
concentration.
Working document QAS/07.242
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Many common bacteriostatic agents (for example, benzyl alcohol) are gradually
destroyed by the effect of radiation in aqueous solutions. The rate of destruction is
dependent upon a number of factors, including the nature of the radionuclide and the
radioactive concentration of the solution. It is therefore not always possible to prescribe
an effective bacteriostatic agent for an injection of a radiopharmaceutical and for certain
preparations the addition of an agent is undesirable; for this reason the inclusion of
bacteriostatic agents is not mandatory. The nature of the bacteriostatic agent, if present,
must be stated on the label; if no bacteriostatic agent is present, this must also be stated.
Radiopharmaceuticals whose expiry periods are greater than one day and that do not
contain a bacteriostatic agent should preferably be supplied in single-dose containers and
if not they should be used within 24 hours after withdrawal of the first dose. Essentially
they are permitted for immediate use only.
Requirements
Identity tests
In general one or two Identification tests have been stated which could involve
determination of radioactive decay, measurement of half-life and determination of the
nature and energy of the radiation.
Identification Method
The following procedure is used for the identification test in “Natrii Phosphatis (32P)
Injectio” for the measurement of beta activity and for calculation of the absorption
coefficient of half-thickness:
Place the radioactive substance, suitably mounted for counting, under a suitable counter.
Make count rate determinations individually and successively, using at least 6 different
thicknesses of aluminium foil chosen from a range of 10 to 200 mg/cm2 and a single
absorber with a thickness of at least 800 mg/cm2. The sample and absorbers should be as
close as possible to the detector in order to minimize scattering effects. Obtain the net
beta count rate at the various absorbers used by subtracting the count rate found with the
thickest absorber (800 mg/cm2 or more). Plot the logarithm of the net beta count rate as a
function of the total absorber thickness. The total absorber thickness is the thickness of
the aluminium plus the thickness of the counter window (as stated by the manufacturer),
plus the air-equivalent thickness (the distance, expressed in cm, of the sample from the
counter window multiplied by 1.205), all expressed in mg/cm2. A linear plot results
approximately.
Choose two of the absorber thicknesses (tl and t2) that are at least 20 mg/cm2 apart and
calculate the absorption coefficient (µ) using the equation.
Working document QAS/07.242
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Where tl is the thinner absorber, t2 is the thicker absorber, Atl and At2
represent the net
beta count rate with tl and t2 absorbers, respectively. Alternatively the half-thickness may
be read directly from the plot.
The choice of absorber depends on the radionuclide. For radionuclides other than
phosphorus-32, which have higher or lower beta energy, greater or lesser absorber
thicknesses are necessary.
For characterization of the radionuclide, the absorption coefficient or the half-thickness
should be within ±5 % of that found for a sample of the same radionuclide of known
radionuclide purity.
The count rate at zero total absorber thickness may be determined by plotting a curve
identical with the one described for determination of the absorption coefficient and
extrapolating the straight line plot to zero absorber thickness, taking into consideration
the thickness, expressed in mg/cm2, of sample coverings, the air, and the counter endwindow.
Radioactive decay
Radioactivity decays at an exponential rate with a decay constant characteristic of each
radionuclide. The curve of exponential decay curve is described mathematically by the
equation:
where N is the number of atoms at elapsed time t, No is the number of atoms when t = 0,
and λ is the disintegration constant characteristic of each individual radionuclide. The
half-life period is related to the disintegration constant by the equation:
The physical half-life of a radionuclide (T½) is the time in which the amount of
radioactivity decreases to one half of its original value. Although the time of decay of an
individual atom can not be determined, large numbers of atoms will obey statistical
considerations and calculations of activity versus time can be carried out. The rate of
decay for a collection of atoms (N) of the same radionuclide is constant and characteristic
for each individual radionuclide.
The radionuclide is generally identified by its half-life or by the nature and energy of its
radiation or by both as stated in the monograph.
Working document QAS/07.242
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Half-life period measurement
The preparation to be tested should under go tested after appropriate dilution to avoid
dead time losses using an ionization chamber, a Geiger-Muller counter, a scintillation
counter or a semiconductor detector. The activity must be sufficiently high to allow
detection during several estimated half-lives. The measured half life should not deviate
more than 5% from half life stated in the Ph. Int.
Determination of Radionuclide Purity
For gamma emitters the most useful method of examination for radionuclide purity is
gamma spectrometry. It is not, however, a completely fool proof method, because:
•
•
•
beta-emitting impurities are, in general, not detected;
When sodium iodide detectors are employed, the photoelectric peaks due to
impurities may be obscured by those due to the major radionuclide, or, in other
words, the degree of resolution of the instrument could be insufficient. This
problem could be solved by the use of high resolution solid state semiconductor
detectors, such as high purity germanium (HPGe) detector.
Unless the instrument has been calibrated with a standard source of known
radionuclide purity under identical conditions of geometry, it is difficult to
determine whether additional peaks are due to impurities or whether they result
from such secondary effects as backscatter, coincidence summation, or
fluorescent X-rays.
The range of gamma spectrometry may be extended in two ways first, by observing
changes in the spectrum of a preparation with time (this is especially useful in detecting
the presence of long-lived impurities in a preparation of a short-lived radionuclide);
secondly, by the use of chemical separations, whereby the major radionuclide may be
removed by chemical means and the residue examined for impurities, or whereby specific
impurities may be separated chemically and then quantified. It is evident that chemical
means will not separate an impurity that is isotopic with the major radionuclide.
Radionuclide impurities are directly related to the production process of a radionuclide.
Based on technical limitations and safety requirements limits have been set for
radionuclidic impurities in radiopharmaceutical preparations, expressed as a percentage
of the total radioactivity.
For identification of gamma emitters the method of choice is gamma spectrometry. In
order to interpret the energy spectrum of radionuclides it is necessary that the energy
range be calibrated with reference radionuclides of high radionuclidic purity (standards).
Gamma spectrometry may be performed using high resolution germanium detectors. Beta
emitting impurities are not detected by gamma spectrometry. Long lived impurities in a
preparation of a short-lived radionuclide may be determined after the decay of the shortlived radionuclide.
Chemical separation of impurities is an effective method both during the production
process and as an analytical procedure. The exact measurement of trace amounts of beta-
Working document QAS/07.242
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and alpha-emitting radionuclides in preparations of generally applied gamma
radionuclides requires special techniques. Chemical separation of the radioactive
impurities is used prior to the measurement of non-penetrating radiation.
Methods of analysis
Determination of Radiochemical Purity
Radiochemical purity can be assessed by a variety of analytical techniques such as liquid
chromatography, paper chromatography and thin-layer chromatography and
electrophoresis. Recently many chromatography columns are used for analysing
radiopharmaceuticals. After or during separation, the distribution of radioactivity on the
chromatogram is determined. Different measuring techniques have to be used depending
on the nature of the radiation and the chromatographic technique. The weight of
substance applied to the chromatogram is often extremely small (because of the great
sensitivity of detection of the radioactivity) and particular care has to be taken in
interpretation with regard to the formation of artefacts. As mentioned above, the addition
of carriers (i.e. the corresponding non-radioactive compounds) for both the
radiopharmaceutical itself and the suspected impurities is sometimes helpful. There is,
however, a danger that when an inactive carrier of the radiopharmaceutical is added it
may interact with the radiochemical impurity, leading to underestimation of these
impurities. In cases where simple chromatographic methods fail to characterize the
labelled compound satisfactorily, high performance liquid chromatographic (HPLC)
could be useful. In some cases the biological distribution of radiopharmaceutical in
suitable test animal can be unavoidable.
Thin-layer chromatography of common radiopharmaceuticals
Radiopharmaceutical
14
C-urea
123/131
Stationary
Mobile
Rf
Rf bound
phase
cellulose
phase
butanol-water-acetic acid
(12:5:3)
chloroform-acetic acid (9:1)
ethyl acetate-ethanol (1:1)
10% ammonium acetatemethanol (1:1)
0.1 M citrate buffer pH 5
acetonitrile-water (95:5)
chloroform-methanol (9:1)
free
0
0.6
0.0
0.6
0.1
0.2-0.3
0.0
1.0
1.0
0.0
0.0
0.0
0.6
1.0
0.0
0.7
0.0
0.3
0.0
0.66
I-hippuran
I-MIBG
111
In-DTPA
silica gel
silica gel
ITLC-SG
111
123/131
In-octreotide
F-FDG
123
I- ioflupane
123
I-iomazenil
ITLC-SG
silica gel
ITLC-SG spot
must be dry
silica gel
123
I-iomazenil
silica gel
131
I-iodocholesterol
silica gel
18
ethyl acetate-ammonium
hydroxide (200:1)
chloroform-acetic acid- water
(65:35:5)
chloroform-ethanol (1:1)
Working document QAS/07.242
page 13
Radiochemical Purity Measurement Systems of Radiopharmaceuticals Thin-layer
chromatography of technetium radiopharmaceuticals
Stationary phases:
ITLC-SG
Instant thin-layer chromatography, silica gel, e.g. Gelman
ITLC-SA
Instant thin-layer chromatography, silicic acid
3MM
Whatman 3MM chromatography paper
No 1
Whatman No 1 chromatography paper
silica gel
Silica gel 60, e.g. Merck
alumina
aluminium oxide, e.g. Bakerflex
cellulose
cellulose, e.g. Merck
Mobile phases:
butanone = 2-butanone = methyl ethyl ketone = MEK
1 M sodium acetate = 82 mg/mL anhydrous sodium acetate
or 136 mg/ml sodium acetate trihydrate
0.1 M citrate = 21 mg/ml monosodium citrate dihydrate
1 M ammonium acetate = 77 mg/ml ammonium acetate
Mixtures of volatile solvents should be made freshly each day
Thin-layer chromatography of technetium radiopharmaceuticals
Radiopharmaceutical
Stationary
phase
Mobile phase
99m
ITLC-SG
99m
ITLC-SG or
3MM
ITLC-SG
MEK, Acetone or
saline
MEK or acetone
Tc-pertechnetate
Tc-MDP
99m
Tc-MDP
99m
Tc-DTPA
99m
Tc-DTPA
99m
Tc-colloid
99m
Tc-DMSA
Tc-DMSA
99m
99m
Tc-MAA
99m
Tc-pyrophosphate
99m
Tc-pyrophosphate
Tc-HSA
99m
99m
Tc-HSA
ITLC-SG or
3MM
ITLC-SG or
3MM
ITLC-SG or
3MM
3MM
ITLC-SA
ITLC-SG or
3MM
ITLC-SG or
3MM
ITLC-SG
ITLC-SG or
3MM
ITLC-SG
strip should be
pre-saturated
Rf
Rf
Rf
RH-Tc
0.0
TcO4
1.0
Tc-bound
-
0.0
1.0
0.0
1 M sodium acetate
or saline
MEK or Acetone
0.0
1.0
1.0
0.0
1.0
0.0
saline
0.0
1.0
1.0
acetone or saline
0.0
1.0
0.0
MEK or acetone
butanol acidified with
0.3 M HCl
MEK, acetone or
saline
MEK or Acetone
0.0
0.0
1.0
0.9
0.0
0.5
0.0
1.0
0.0
0.0
1.0
0.0
Water
MEK or Acetone
0.0
0.0
1.0
1.0
1.0
0.0
ethanol-ammoniawater (2:1:5)
0.0
1.0
1.0
Working document QAS/07.242
page 14
99m
Tc-HIG
99m
Tc(V)-DMSA
Tc(V)-DMSA
99m
Tc(V)-DMSA
99m
99m
Tc-IDAs
Tc-IDAs
99m
99m
Tc-IDAs
99m
Tc-sestamibi
99m
Tc-tetrofosmin
99m
Tc-MAG3
99m
Tc-MAG3
Tc-exametazime
99m
Tc-exametazime
99m
Tc-exametazime
99m
Tc-sulesmurab
99m
99m
Tc-depreotide
99m
Tc-depreotide
with human
serum albumin
and dried
ITLC-SG or
3MM
ITLC-SG
ITLC-SG
silica gel
ITLC-SA
3MM spot
must be dry
ITLC-SG
Alumina
Pre-spot with
ethanol; do not
allow spot to
dry
ITLC-SG
spot must be
dry
ITLC-SG
ITLC-SG
ITLC-SG
ITLC-SG
No 1
ITLC-SG or
3MM
ITLC-SG
ITLC-SG
acetone, saline, or 0.1
M citrate
butanone
saline
butanol-acetic acidwater (3:2:3)
20% sodium chloride
butanone
0.0
1.0
0.0
0.0
0.0
0.0
1.0
1.0
0.8
0.0
1.0
0.5
0.0
0.0
1.0
0.9
0.0
0.0
water or 50%
acetonitrile
ethanol
0.0
1.0
1.0
0.0
0.0
1.0
acetonedichloromethane
(35:65)
0.0
1.0
0.5
ethyl acetatebutanone (3:2)
50% acetonitrile
butanone
saline
50% acetonitrile
acetone, saline, or 0.1
M citrate
saturated solution of
sodium chloride
1 M ammonium
acetate-methanol
(1:1)
0.0
1.0
0.0
0.0
0.0
0.0
0.0
0.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.0
1.0
0.0
0.0
1.0
0.0
0.0
1.0
1.0
Substitutions:
• in most cases, 2-butanone (methyl ethyl ketone, MEK) can be substituted for
acetone
• in most cases, water can be substituted for saline
• in most cases, Whatman No 1 can be substituted for Whatman 3MM paper
• ACD can be substituted for 0.1 M citrate
Solid phase separation and radiopharmaceuticals
The use of solid phase extraction cartridge methods although expensive is becoming
increasingly common.
General procedure:
1. Pre-wet (“activate”) cartridge with 2-5ml ethanol or methanol.
Working document QAS/07.242
page 15
2. Prepare cartridge with 2-10ml of preparation solvent.
3. Place a drop of the radiopharmaceutical in the inlet of the cartridge.
4. Elute sequentially with 2-10ml quantities of elutes A, B, C and collect each in a
separate tube; after the last elute, force air through the cartridge to dry it.
5. Place the cartridge in another tube for measurement of residual activity.
6. Measure the activity in each tube in an ionisation chamber.
7. Calculate radiochemical purity as per table.
Radiopharmaceutical
Type of
cartridge
alumina
N
C18
Preparation
solvent
0.5 ml ethanol
A
B
10ml ethanol
cartridge residue
2ml saline
2ml saline
5ml ethanol
99m
C18
2ml saline
2ml saline
5ml ethanol
99m
silica
5ml saline then
1ml air
10ml methanol-water
(70:30) over 2 minutes
cartridge residue
99m
silica
5ml saline then
1ml air
10ml methanol-water
(70:30) over 2 minutes
10ml methanolsaline (80:20)
cartridge
residue
B/total
99m
C18
10ml
10ml
10ml 50%
ethanol
cartridge
residue
B/total
1 mM HCl
1 mM
5ml 0.5%
ethanol in PB
10ml 7%
ethanol
in PB
99m
Tc-sestamibi
99m
Tc-sestamibi
Tc-tetrofosmin
Tc-tetrofosmin
Tc-tetrofosmin
Tc-MAG3
99m
C18
10ml
HCl
5ml
99m
Tc-exametazime
99m
Tc-exametazime
C18
C18
1 mM HCl
5ml. saline
5ml saline
1 mM HCl
5ml saline
5ml saline
cartridge residue
5ml ethanol
111
In-octreotide
C18
10ml water
5ml water
5ml methanol
123
I-ioflupane
C18
5ml water
5ml water
5ml ethanol
10ml PB-THF
(3:1)
cartridge residue
Tc-MAG3
123/131
I-MIBG
C18
5ml water
5ml water
123/131
I-MIBG
C18
5ml water
5ml 10 mM NaOH
C
D
Purity
A/total
cartridge
residue
cartridge
residue
B/total
B/total
B/total
cartridge
residue
cartridge
residue
cartridge
residue
cartridge
residue
cartridge
residue
Prepare 100ml 0.01M monosodium phosphate solution (NaH2PO4). Prepare 20ml 0.01 M
disodium phosphate solution (Na2HPO4). Add 10ml disodium phosphate solution to
100ml monosodium phosphate solution. pH should still be below 6. Add disodium
phosphate solution drop wise until pH of 6 is obtained.
Preparation of reagents
1 mM HCl (0.001 M HCl) = 1ml conc. HCl per litre of distilled water. PB for MAG3 =
0.01 M (10 mM) sodium phosphate buffer pH 6. PB for MIBG = 0.1 M (100 mM)
monosodium phosphate (NaH2PO4). THF = tetrahydrofuran10 mM NaOH (0.01 M
C/total
B/total
B/total
B/total
B/total
B/total
B/total
Working document QAS/07.242
page 16
NaOH) = 0.4 g dissolved in 1 litre of distilled water or dilute 1ml 1 M NaOH with 99ml
distilled water. Cartridges can be re-used after decay of radioactivity.
HPLC methods for Radiopharmaceuticals
Radiopharmaceutical
99m
C-8
Isocratic
or gradient
gradient
99m
C-18
isocratic
99m
PRP-1
gradient
99m
PRP-1
isocratic
99m
PRP-1
isocratic
99m
C-18
isocratic
with wash
99m
C-18
gradient
99m
PRP-1
gradient
99m
PRP-1
gradient
99m
PRP-1
gradient
99m
C-18
gradient
123/131
I-MIBG
C-18
isocratic
123
I-ioflupane
C-18
isocratic
123
I-iomazenil
C-18
isocratic
Tc-sestamibi
Tc-sestamibi
Tc-tetrofosmin
Tc-tetrofosmin
Tc-tetrofosmin
Tc-MAG3
Tc-MAG3
Tc-exametazime
Tc-exametazime
Tc-exametazime
Tc-depreotide
Column
Solvent(s)
A: 50 mM ammonium sulphate
B: methanol
0%B to 95%B over 5 minutes
A: methanol
B: 50 mM ammonium sulphate
C: acetonitrile
A:B:C 45:35:20
A: 10 mM phosphate buffer pH 7.5
B: tetrahydrofuran
0%B to 100%B over 17 minutes
A: acetonitrile
B: 10 mM ammonium carbonate
A:B 70:30
A: 5 mM monopotassium phosphate
B: acetonitrile
A:B 50:50
A: ethanol
B: 10 mM phosphate buffer pH 6
A:B 5:95
after peak, wash with methanol-water 90:10
A: 10 mM potassium phosphate with 1%
triethylamine pH 5
B: tetrahydrofuran
0%B to 8%B over 30 minutes
A: 20 mM phosphate buffer pH 7.4
B: tetrahydrofuran
0%B to 25%B over 6 minutes
A: 10 mM potassium phosphate pH 7 or water
containing 1% methanol
B: acetonitrile
0%B to 50%B over 5 minutes
A: 50 mM sodium acetate pH 5.6
B: tetrahydrofuran
0%B to 100%B over 17 minutes
A: 0.1% TFA in water
B: 0.1% TFA in acetonitrile
20%B to 27%B over 30 minutes
A: 100 mM sodium phosphate
B: tetrahydrofuran
A:B 88:12
A: methanol
B water
C: triethylamine
A:B:C 85:15:0.2
A: methanol
B: water
A:B 55:45
Working document QAS/07.242
page 17
125
I-albumin
C-4
gradient
111
In-octreotide
C-18
gradient
amino
isocratic
18
F-FDG
A: 0.1% TFA in water
B: 0.1% TFA in acetonitrile
35%B to 90%B in 10 minutes
A: saline
B: methanol
40%B to 80%B in 20 minutes
A: acetonitrile
B: water
A:B 95:5
Determination of Chemical Purity
Chemical purity refers to the proportion of the preparation that is in the specified
chemical form regardless of the presence of radioactivity; it may be determined by
accepted methods of analysis.
The chemical purity of a preparation is often no guide to its radiochemical purity.
Preparations, especially those resulting from exchange reactions (for example, a
preparation of o-iodohippuric acid in which some of the iodine atoms are replaced by
atoms of iodine-131), may be of high chemical purity but may contain impurities of high
specific activity (that is, a tiny weight of an radiochemical impurity may be associated
with a relatively large amount of the radionuclide).
In general, chemical impurities in preparations of radiopharmaceuticals are objectionable
only if they are toxic or if they modify the physiological processes that are under study or
if they result in undesirable interactions (e.g. aluminium can induce flocculation of Tc99m sulphur colloid). Special attention is necessary for substances with a
pharmacologically active or pharmacodynamic effect even for very low amounts (i.e.
receptor ligands). Where appropriate, the stereo-isomeric purity has to be verified.
General limits concerning arsenic and heavy metal contents in pharmaceutical
preparations are valid for radiopharmaceuticals as well (see Ph. Int.)
pH For routine radiopharmaceutical practices
The Ph. Int. outlines standard methods for analysis of pH and these should be used for all
non-radioactive solution. For radioactive solutions however due to risk of high radiation
exposure and limited quantity of solution it is common to find use of strip pH paper.
However it is essential any pH strips used to be properly validated and checked against
buffers. This validation is critical at times such as change of supplier of pH strips. It is
encouraged that in general routine validation and comparison are done using nonradioactive buffers.
Electrophoresis and RP
The Ph. Int. outlines standard methods for analysis using electrophoresis techniques and
these in the main can also be used for radiopharmaceuticals. Suitable counting devices
and detectors are the only additional requirements. These methods are particularly
suitable for charged radiopharmaceuticals (anionic, e.g. HIDA complexes, radioiodinated
o-hippuric acid, MAG3 or cationic, e.g. MIBI).
Working document QAS/07.242
page 18
Tin analysis
Tin is used for many technetium based radiopharmaceuticals and since this is the main
radiopharmaceutical that is most widely used clinically the assessment of tin is essential.
For an optimal radiopharmaceutical formulation milligram amounts are used and for
some microgram are used. The actual levels can affect the final radiochemical purity and
alter the pharmacokinetics of the radiopharmaceutical. Well-established methods are
identified and used as the standard methods of analysis for tin estimation. Many of the
analytical methods used for environmental samples are the methods approved by Federal
agencies and organizations such as EPA (Environmental Protection Authorities) and the
National Institute for Occupational Safety and Health (NIOSH). Other methods are those
that are approved by groups such as the Association of Official Analytical Chemists
(AOAC) and the American Public Health Association (APHA). Additionally, analytical
methods are included that modify previously used methods to obtain lower detection
limits and/or to improve accuracy and precision. Individual monographs will contain
specific requirement.
Tin estimation by Gas Chromatography (GC) or HPLC
Tin is usually determined as the total metal, but it may also be measured as specific
organo-tin compounds. Flame atomic absorption analysis is the most widely used and
straightforward method for determining tin; furnace atomic absorption analysis is used
for very low analyte levels and inductively coupled plasma atomic emission analysis is
used for multi-analyte analyses that include tin. The preferred separation technique for
organo-tin compounds is gas chromatography (GC) due to its high resolution and detector
versatility. High performance liquid chromatography (HPLC) has also been used in the
analysis of organo-tin compounds. The advantage of HPLC over GC is that no
derivatization step is needed after extraction.
For determination of tin in biological samples, the sample is digested in an oxidizing acid
mixture followed by atomic spectrometric determination. Determination of organo-tin
compounds in biological materials will require extraction, derivatization, separation, and
detection, as described. Whole blood samples are typically analysed by
spectrophotometry and photometry.
Tin estimation by polarography
Tin can be effectively analysed by polarography, which is also called polarographic
analysis, or voltammetry method of analysing solutions of reducible or oxidizable
substances. Polarography technique involves electric potential (or voltage) varied in a
regular manner between two sets of electrodes (indicator and reference) while the current
is monitored. The shape of a polarogram depends on the method of analysis selected, the
type of indicator electrode used, and the potential ramp that is applied. The method is
useful in detecting several substances simultaneously and is applicable to relatively small
concentrations, e.g. 10-6 up to about 0.01 mole per litre, or approximately 1 to 1000 parts
per million.
Working document QAS/07.242
page 19
Tin estimation by Potentiometric titration with standard potassium iodate (KIO3)
solution
Potentiometric titration is based on the principle of measuring the change in redox
potential when tin solution is titrated against potassium iodate solution. The redox
potential is measured with redox-electrode couple. This method is ideal for estimating
stannous (tin II) contents in radiopharmaceutical vials sealed in Nitrogen or inert gases.
Tin estimation is not possible in vials containing antioxidants such as ascorbic acid or
gentisic acid. Since antioxidants are commonly found in radiopharmaceuticals this
method is not suitable for such formulations.
Reagents
The following two reagents are prepared as described: KIO3 (A): Preparation of
1.667x10-3 M solution (Stock solution): Approx. 200 mg potassium iodate (AR Grade) is
dried in an oven at 120oC for 1 hour and allowed to cool in a desiccator. Exactly 89.18
mg is weighed out and dissolved in 250ml water for irrigation, N2 purged before use. A
fresh solution is prepared every three months. KIO3 (B): Preparation of 3.334x10-4M
solution (Working solution): 10ml KIO3 (A) is diluted to 50ml with water for irrigation
and purged with N2 for 5 minutes. This solution is to be prepared every day.
Titration method
The set up consists of titration cell assembly with redox-electrode (e.g. Metrohm)
operating in milli-volt mode. A gentle stream of N2 is passed through the assembly to
mix the solution and provide an inert atmosphere. A stannous containing vial (e.g. PYP)
is reconstituted with 4.0ml of saline for injection. 1.0ml of the solution is dispensed into
the titration cell. 2.0ml of 1M hydrochloric acid (HCl) is added into the cell. It is titrated
immediately with standard KIO3 using a microburette until a marked, persistent jump in
redox-electrode potential is achieved. The volume of potassium iodate required to
neutralize tin is “The end-point”.
Calculation
Radiopharmaceutical kits containing high stannous (tin II) contents (e.g. PYP and Phytate
colloid kits) are titrated with KIO3 (A) solution which contains 594 microgram Sn(II)per
ml solution. Projected end-point is indicated.
Theoretical Sn(II) KIO3 (A)
content /ml
(end-point)
PYP
2000
3.37
PHYTATE
532
0.90
Working document QAS/07.242
page 20
Radiopharmaceutical kits containing low stannous (tin II) contents (e.g. DTPA, DISIDA
kits) are titrated with KIO3 (B) solution, which contains 119 microgram Sn(II) in 1.0ml.
Projected end-point is indicated.
Theoretical Sn(II) ml of KIO3 (B)
(end-point)
content /ml)
DTPA
313
2.61
DISIDA
313
2.61
IDP
263
2.20
SnF2
488
4.11
Pass criteria
In general, radiopharmaceutical kits containing more than 85% of the theoretical content
of tin (II) is an acceptable “pass Criteria”. See specific pass criteria of individual
monograph.
Tests for Sterility
A number of monographs for radiopharmaceuticals contain the requirement that the
product be sterile and free of endotoxins. Special difficulties arise with
radiopharmaceutical preparations because of the short half life of most radionuclides,
small size of batches and the radiation hazards. The half-life of many
radiopharmaceuticals is so short that the sterility test is initiated and bacterial endotoxins
test completed prior to release. The tests must in such cases be completed retrospectively.
The manufacturer should begin the sterility test as soon as possible and read the results
after release. A particular responsibility falls upon the manufacturer of
radiopharmaceuticals to validate the sterilization process by all suitable measures, which
may include careful and frequent calibration of sterilizers and the use of biological and
chemical indicators of the efficiency of the sterilization process. See more details in Ph.
Int. chapter.
Sampling
Number of containers
Minimum number of samples
in the batch
to be tested
not more than 100
10% or 4 containers (whichever is greater)
between 100 and 500
10 containers
more than 500
2% or 20 containers (whichever is less)
Working document QAS/07.242
page 21
For liquids
Quantity in the container
Quantity of sample needed
less than 1 ml
entire contents of container
between 1 ml and 4 ml
half contents of container
between 4 ml and 20 ml
2 ml
20 ml or more (including largevolume parenterals)
10% of contents
When the size of the batch of a radiopharmaceutical is limited to one or few samples (e.g.
therapeutic or very short-lived radiopharmaceutical preparations), sampling the batch
may not be possible. The parametric release of the product manufactured by a fully
validated process is then the method of choice. When the half-life is very short (e.g. less
than 20 minutes), the administration of the radiopharmaceutical to the patient is generally
on-line with a validated production system.
The Ph. Int. Sterility methods using membrane filtration or direct inoculation test are
most preferred.
Sterility Incubation
Incubate portions of the media at temperatures 30-35°C if it is intended to detect mainly
bacteria and at 20-25°C if it is intended to detect fungi for not less than 14 days. No
growth of microorganisms occurs.
Bacterial endotoxins and Pyrogen tests
The manufacturer also bears a particular responsibility to ensure that all substances used
in the preparation of radiopharmaceuticals are handled in a manner that ensures their
freedom from pyrogens. These tests are specified in certain monographs see Ph. Int.
sections and mainly applicable mainly applicable to final products with the injection
volume larger than 15ml. Prevalidation of the test is recommended to exclude any
interference or artefact due to radiopharmaceutical.
The method for the detection of Gram-negative bacterial endotoxins is based on the
gelation of a lysate of amoebocytes (limulus amoebocyte lysate, LAL) from the
horseshoe crab, Limulus polyphemus or Limulus tachypleus. The addition of a solution
containing endotoxins to a solution of the lysate produces turbidity, precipitation, or
gelation of the mixture. The rate of reaction depends on the concentration of endotoxin,
the pH, and the temperature. The reaction requires the presence of certain divalent cations,
an enzyme system, and protein capable of clotting, which are provided by the lysate. The
pH of some radiopharmaceuticals will require to be adjusted to pH 6.5-7.5 to achieve
optimal results. The levels of radioactivity should be standardized as some types of
radioactivity and radionuclides especially high levels of activities can interfere with these
tests.
Working document QAS/07.242
page 22
Biodistribution
Selection of species
A physiological distribution test is prescribed, if necessary, for certain
radiopharmaceutical preparations. Specific requirements are set out in individual
monographs. The distribution pattern of radioactivity observed in specified organs,
tissues or other body compartments of an appropriate animal species (usually rats or mice)
can be a reliable indication of the expected distribution in humans and thus of the
suitability of the intended purpose. The individual monograph prescribes the details
concerning the performance of the test and the physiological distribution requirements,
which must be met for the radiopharmaceutical preparation. A physiological distribution
conforming to the requirements will assure appropriate distribution of the radioactive
compounds to the intended biological target in humans and limits its distribution to nontarget areas.
Unless otherwise directed, animals used in an assay or tests are healthy animals, drawn
from uniform stocks that have not previously been treated with any material that will
interfere with the test. If relevant, the species, sex, strain and weight and/or age of the
animals are specified in the monograph. Unless otherwise stated, mice weigh not less
than 20g and not more than 30g; rats weigh not less than 150g and not more than 250g;
and guinea pigs (especially for cardiac radiopharmaceuticals) weigh not less than 250g,
or when used in systemic toxicity tests, not less than 350g.
In general, the radiopharmaceutical preparation intended for human use is injected in at
least three animals. The administration will normally be made via the intravenous route
(i.v.) for which purpose the caudal vein is used. Other veins such as the saphenous,
femoral, jugular or penile veins may be used in special cases. Animals showing evidence
of extravasations of the injection (observed at the time of injection or revealed by
subsequent assay of tissue radioactivity) are rejected from the test. In the case of
lymphatic mapping agents (e.g. antimony sulphide colloid, nano-colloid), rabbit or rat
may be injected with the radiopharmaceutical by subdermal injection between the webs
of the hind toes. Where applicable, products are reconstituted according to the
manufacturer’s instructions. In most cases, dilution immediately before injection may be
necessary to ensure optimal radioactivity count characteristics.
Mice should be warmed to room temperature under an IR (Infrared) lamp before injection
of radiopharmaceutical dose (x). Swab the injection site with cotton wool, which should
be saved for counting (y) and the residual dose in the syringe after injecting should also
be counted (z).
Actual Injected dose (a)= x-(y+z).
Immediately after injection, each animal should be placed in a separate cage, which will
allow collection of excreta and prevent contamination of the body surface of the animal.
Working document QAS/07.242
page 23
After X hour, sacrifice the animal by an ethical method, including carbon dioxide
asphyxiation. Time varies for individual tests. Collect sample of blood by cardiac
puncture and note the weight. Normally blood is approx. 7% of total body weight. Dissect
out required organs and tissues, e.g. gall bladder, liver, stomach, intestines, bones and
kidneys and place in labelled counting tubes. Remove tail above injection site and place
in counting tube. Prepare three dose standards (0.2ml) in counting tubes. Count remaining
organs and standards in an automatic gamma-well counter or other suitable devices.
Determine the percentage of injected radioactivity in all organs according to the
following formula: 100 x (A/B) where: A = radioactivity in organ; B = injected
radioactivity.
The percentage of radioactivity in blood is determined according to the formula:
[100x(C/Ws) x 0.07 x (Wr)] / B
where C = Radioactivity in specimen of blood; Ws = weight in grams of blood
specimen and Wr = weight in grams of mouse.
Note: The physiological distribution is then calculated and expressed as the percentage
of the injected dose/gram wet weight of tissue. Tissues are counted in optimally
calibrated gamma counters.
Specification (Pass Criteria)
A radiopharmaceutical should satisfy certain specification/s (pass criteria) before it is
released for human use
1. Not less than X% of the injected dose is found in the “Target” organ. Not more
than Y% is present in the non-Target organ. Not more than Z% is present in the
excretory organ (e.g. kidneys). Not more than S% is present in the stomach. Not
more than B% is present in the blood.
2. For a preparation to meet the requirements of the test, the distribution of
radioactivity in at least two of the three animals must comply with all the
specified criteria.
Biodistribution of individual, commonly used radiopharmaceuticals
Biodistribution study: 99mTc-DISIDA [2,6-dimethylphenylcarbamoylmethyl
iminodiacetic acid)
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For these
particular test Balb/c female mice, weighing 20-30g are used.
The kit should be reconstituted with 2MBq of 99mTc04- in 5.0ml of 0.9% w/v saline. Wait
approximately 1 hour before injecting. Inject 0.2ml (approximately 80kBq) into caudal
vein of three animals and wait for localization time for 1.0 hour.
Working document QAS/07.242
page 24
The mice are warmed to room temperature under IR lamp before RP injection. Save
cotton wool swab for counting. Place in a beaker containing tissue and cover with a wire
grid. After 1 hour, sacrifice the animal by carbon dioxide asphyxiation. Collect sample of
blood by cardiac puncture and place in a tarred counting tube. Dissect out gallbladder,
liver, stomach, intestines and kidneys and place in labelled counting tubes. Remove tail
above injection site and place in counting tube. Prepare three dose standards (0.2ml) in
counting tubes. Count remaining organs and standards in an automatic gamma-well
counter. Determine the percentage of injected radioactivity in all organs including blood
according to earlier section.
Specification or pass criteria
Not less than 70.0% of the injected dose is found in the gallbladder and intestines. Not
more than 10.0% is present in the liver. Not more than 10% is present in the kidneys.
Not more than 3.0% is present in the stomach. Not more than 3.0% is present in the
blood.
Biodistribution study: 99mTc- Succimer (DMSA [Dimercaptosuccinic acid] )
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For this
particular test, albino male rats, weighing 150-250g needs to be used.
The kit should be reconstituted with 1MBq of 99mTc04- in 5.0ml of 0.9% w/v saline. Wait
approximately 1 hour before injecting. Inject 0.2ml (approximately 40kBq) into caudal
vein of three animals and wait for localization time for 1.0 hour.
The rats were warmed to room temperature under IR lamp and inject dose. Save cotton
wool swab for counting. Place in a beaker containing tissue and cover with a wire grid.
After 1 hour, sacrifice the animal by placing them in a CO2 chamber. Dissect out various
organs [e.g. kidneys, liver, stomach and lungs] and place them in labelled counting tubes.
Remove tail above injection site and place in counting tube. Prepare three dose standards
(0.2ml) in counting tubes. Count remaining organs and standards in an automatic gammawell counter. The percentage of injected radioactivity in all organs including blood was
determined as described in the introduction.
Specification or pass criteria
Not less than 40.0% of administered radioactive dose is found in the kidneys, not more
than 10.0% in the liver, not more than 2.0% in the stomach and not more than 5.0% in the
lungs.
Biodistribution study: 99mTc- Pentetate (DTPA [diethylenetriaminepentaacetic acid])
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For this
particular test Balb/c female rats, weighing 150-250g should be used.
Working document QAS/07.242
page 25
The kit should be reconstituted with 10MBq of 99mTc04- in 5.0ml of 0.9% w/v saline. Add
1.0ml of this solution to 9.0ml of 0.9%w/v saline purged with nitrogen gas. Wait
approximately 1 hour before injecting. Inject 0.2ml (approximately 50kBq) into caudal
vein of three animals and wait for localization time for 1.0 hour.
The rats were warmed to room temperature under IR lamp and inject dose. Save cotton
wool swab for counting. Place in a beaker containing tissue and cover with a wire grid.
After 1 hour, sacrifice the animals by placing them in a CO2 chamber. Swab any urinecontaminated fur with damp tissue and add to the beaker. Dissect out liver, stomach,
intestines and kidneys and place in labelled tubes. Remove tail above injection site and
place in counting tube. Prepare three dose standards (0.2ml) in counting tubes. Count
remaining organs and standards in an automatic gamma-well counter or other suitable
devices.
Prepare a urine standard by adding 0.2ml urine into a 20ml tube containing 10ml water.
Add 10ml water to each urine beaker and transfer wet tissue to a counting tube. Wipe the
beaker with tissue and add to the counting tube. Rinse the beaker with a further 2 x 10 ml
and transfer to counting tubes. Prepare organ standards by adding 0.2ml into each of three
counting tubes. Count all samples in a gamma counter and determine the % of
radioactivity in the urine and organs by comparison with the relative standards according
to method described in the introduction.
Specification or pass criteria
At 2 hours post injection the sum of the percentages of radioactivity found in urine and
bladder should be more than 85% of injected radioactivity. Less than 1% of injected
activity should be found in liver.
Biodistribution study: 99mTc- Methylene diphosphonate [MDP]
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For this
particular test, albino male rats, weighing 150-250 g should be used.
The kit should be reconstituted with 1 MBq of 99mTc04 - in 5.0ml of 0.9% w/v saline. Add
0.5ml of this solution to 9.5ml of 0.9% w/v saline purged with nitrogen gas. Wait
approximately 1 hour before injecting. Inject 0.2ml (approximately 20kBq) into caudal
vein of three animals and wait for localization time for 2.0 hours.
The rats were warmed under IR lamp and inject dose. Save cotton wool swab for
counting. Place in a beaker containing tissue and cover with a wire grid. After 2 hours,
sacrifice the animals by placing them in a CO2 chamber. Place samples of blood, muscle
and the two femurs in tarred counting tubes. Dissect out the liver and place in a labelled
counting tube. Remove tail above injection site and place in counting tube. Prepare three
dose standards (0.2ml) in counting tubes. Count remaining organs and standards in an
automatic gamma-well counter and calculate the % injected dose/gram wet weight in the
femurs and liver as described in the introduction. Calculate the ratios: % Dose/g in
femurs: % Dose/g muscle and % Dose g in femurs: % Dose/g blood.
Working document QAS/07.242
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Specification or pass criteria
In not fewer than two of the three rats not less than 2.5% of the radioactivity is present in
the femurs, not more than 1% is present in the liver, the ratio A1/A2 is not less than 100
and the ratio A1/A3 is not less than 40. The radioactivity per gram weight is represented
in the femora (A1), muscle (A2) and blood (A3).
Biodistribution study: 99mTc- Pyrophosphate [PYP]
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For this
particular test, albino male rats, weighing 150-250g should be used.
Dilute multidose kit to 5.0ml with 0.9% w/v saline and transfer 1.0ml to a nitrogen-filled
10ml vial. Add 20 MBq of 99mTcO4- in 4.0ml of 0.9%w/v saline. Add 1.0 ml. of this
solution to 9.0ml of 0.9%w/v saline purged with nitrogen gas. Inject 0.2ml
(approximately 0.4 MBq) into caudal vein of three animals and wait for localization time
for 2.0 hours.
The rats were warmed to room temperature under IR lamp and inject dose. Place in a
beaker containing tissue and cover with a wire grid. After 2 hours, sacrifice the animals
by placing them in a CO2 chamber. Place samples of blood, muscle and the two femurs in
tarred counting tubes. Dissect out the liver and kidneys and place in labelled counting
tubes. Remove tail above injection site and place in counting tube. Prepare three dose
standards (0.2ml) in counting tubes. Count remaining organs and standards in an
automatic gamma-well counter and calculate the % injected dose/gram wet weight in the
femurs and liver. Calculate the ratios: % Dose/g in femurs: % Dose/g muscle and % Dose
g in femurs: % Dose/g blood.
Specification or pass criteria
In not fewer than two of the three rats not less than 2.5% of the radioactivity is present in
the femurs, not more than 1% is present in the liver, the ratio A1/A2 is not less than 100
and the ratio A1/A3 is not less than 40. The radioactivity per gram weight is represented
in the femora (A1), muscle (A2) and blood (A3).
Biodistribution study: 99mTc- Tin colloid (Stannous colloid)
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For these
particular test Balb/c female mice, weighing 20-30 g should be used.
Prepare stannous colloid according to the standard with 1MBq of 99mTcO4- in a total
volume of 3.0ml. Wait before injecting 0.2ml (approximately 70kBq) into caudal vein of
three animals and wait for localization time for 15 minutes.
The mice were warmed to room temperature under IR lamp and inject dose. Place in a
beaker containing tissue and cover with a wire grid. After 15minutes, sacrifice the
Working document QAS/07.242
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animals by placing them in a CO2 chamber. Place samples of blood, muscle and the two
femurs in tarred counting tubes. Dissect out liver, spleen and lungs and place in labelled
counting tubes. Remove tail above injection site and place in counting tube. Prepare three
dose standards (0.2ml) in counting tubes. Count remaining organs and standards in an
automatic gamma-well counter and calculate the % injected dose/gram wet weight in the
liver, spleen and lungs as described in introduction.
Specification or pass criteria
Not less than 80.0% of the radioactivity is found in the liver and spleen and not more than
5.0% of the radioactivity is found in the lungs, in each of the three mice. A repeat test
may be performed, in which case five of the six mice must comply with the specification.
Biodistribution study: 99mTc- Antimony sulphide colloid
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. For these
particular test Balb/c female mice, weighing 20-30g should be used.
Prepare 99mTc antimony sulphide colloid according to the standard protocol. Inject 0.1ml
(approximately 2MBq) into caudal vein of three animals and wait for localization time of
approximately 20 minutes.
The mice were warmed to room temperature under IR lamp and inject dose. Place in a
beaker containing tissue and cover with a wire grid. After 15minutes, sacrifice the
animals by placing them in a CO2 chamber. Dissect out liver, spleen and lungs and place
in labelled counting tubes. Remove tail above injection site and place in counting tube.
Prepare three dose standards (0.2ml) in counting tubes. Count remaining organs and
standards in an automatic gamma-well counter and calculate the % injected dose/gram
wet weight in the liver, spleen and lungs as described in the introduction.
Specification or pass criteria
Not less than 80.0% of the radioactivity is found in the liver and spleen and not more than
5.0% of the radioactivity is found in the lungs, in each of the three mice.
Qualitative results (optional)
Inject a rabbit (rat may be used) with 20-50MBq of [99mTc] Antimony Sulphide colloid,
subdermally between the web of the hind toes. Record distribution on scintiphotos 60
minutes after injection. The images should show migration along the lymphatics and
localization in regional lymph nodes.
Biodistribution study: 99mTc-Exametazime [HMPAO]
Record the details of manufacturer, batch number, expiry date and date of the study
before performing biodistribution test for a particular radiopharmaceutical. Inject
intravenously between 3MBq and 80MBq of RP, in a volume of 0.2 to 0.25ml, into the
caudal vein of each of three 125g to 225g anesthetized Sprague-Dawley female rats.
Sacrifice the animals 30 minutes after the injection, and carefully remove the main organs
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including the brain. Keep each organ separate organs after dissection. Place each organ
and the remaining carcass (excluding the tail) in separate, suitable counting containers,
and determine the radioactivity, in counts per minute, in each container with an
appropriate detector, using the same counting geometry. Determine the percentage of the
administered radioactive dose in each organ.
Specification or pass criteria
Not less than 1.5% of the radioactivity is found in the brain in each of the three rats.
Working document QAS/07.242
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ANNEX
TERMINOLOGY AND METHODS OF DETECTION AND MEASUREMENT
Nuclide
A unique atom characterized by its atomic number (number of protons in the nucleus)
and its atomic mass number (total number of neutrons and protons in the nucleus) and
having stability such that its lifetime is measurable. All atoms sharing the same atomic
number are the same element.
Isotopes
Atoms of the same element with different atomic mass numbers are called isotopes.
Radionuclide
A nuclide that is unstable and will eventually undergo radioactive decay. Radionuclides
are produced using a nuclear reactor through either nuclear fission or neutron activation
or by the use of a particle accelerator. Radionuclides can also be obtained from a
radionuclide generator. A radionuclide generator is a system incorporating a relatively
long-lived radionuclide called the parent which will be obtained from a nuclear reactor or
accelerator. The parent decays to produce a relatively short-lived radionuclide called the
daughter that is removed from the generator by elution or other procedures and is used in
radiopharmaceutical preparation.
Activity (Radioactivity)
A measure of atoms in a particular sample that undergo radioactive decay within a certain
time period is generally referred to as the activity. The term activity is generally
understood to be synonymous with radioactivity. The term radioactivity should be
employed where there will be any ambiguity since the term “activity” has additional
meanings within the pharmacopoeia.
Radioactivity
The property of certain nuclides of emitting radiation by the spontaneous transformation
of their nuclei into those of other nuclides.
EXPLANATORY NOTE. The term “disintegration” is widely used as an alternative to
the term “transformation”. Transformation is preferred as it includes, without semantic
difficulties, those processes in which no particles are emitted from the nucleus.
Radioactive decay
The property of unstable nuclides during which they undergo a spontaneous
transformation within the nucleus. This change results in the emission of energetic
particles or electromagnetic energy from the atoms and the production of an altered
nucleus.
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EXPLANATORY NOTE. The term “disintegration” is widely used as an alternative to
the term “transformation”. Transformation is preferred as it includes, without semantic
difficulties, those processes in which no particles are emitted from the nucleus.
Units of radioactivity
The activity of a quantity of radioactive material is expressed in terms of the number of
nuclear transformations taking place in unit time. The SI unit of activity is the Becquerel
(Bq), a special name for the reciprocal second (s-1). The expression of activity in terms of
the Becquerel therefore indicates the number of disintegrations per second.
Activity in the SI system is measured in atoms per second undergoing spontaneous
transformation. The unit for 1 transformation per second is the Becquerel (Bq). The
Becquerel replaces the historical unit of activity called the curie. The curie (Ci) is
equivalent to 3.7 x 1010 Bq.
The conversion factors between Becquerel and Curie and its submultiples are given in
Table.
Table. Units of radioactivity commonly encountered with radiopharmaceuticals and the
conversions between Le Système International d'Unités or SI units and Historical Units
Number of atoms
transforming per second
SI Unit: Becquerel (Bq)
Historical Unit: Curie (Ci)
1
1 Bq
27 picoCurie (pCi)
1000
1 kiloBecquerel (kBq)
27 nanoCurie (nCi)
1 x 106
1 megaBecquerel (MBq)
27 microCurie (µCi)
1 x 109
1 gigaBecquerel (GBq)
27 milliCurie (mCi)
37
37 Bq
1 (nCi)
37,000
37 kBq
1 (µCi)
3.7 x 107
37 MBq
1 (mCi)
3.7 x 1010
37 GBq
1 Ci
Detection and Measurement
Radioactive decay may involve the emission of charged particles, the process of electron
capture, or the process of isomeric transition. The charged particles emitted from the
nucleus may be alpha particles (helium nuclei of mass number 4) or beta particles
Working document QAS/07.242
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(electrons of negative or positive charge, beta − or beta + respectively, the latter known as
positrons). The emission of charged particles from the nucleus may be accompanied by
gamma rays, which are of the same physical nature as X-rays. Gamma rays are also
emitted in the process of isomeric transition (IT). X-rays, which may be accompanied by
gamma rays, are emitted in the process of electron capture (EC). Positrons are annihilated
on contact with matter. Each positron annihilated is accompanied by the emission of 2
gamma rays, at 180 degrees to one another, each with energy of 0.511 MeV.
The methods employed for the detection and measurement of radioactivity are dependent
upon the nature and energy of the radiation emitted. Radioactivity may be detected and/or
measured by a number of different instruments based upon the action of radiation in
causing the ionization of gases and solids, or the scintillation in certain solids and liquids,
or by the effect of radiation on a photographic emulsion.
In general, a counting assembly consists of a sensing unit and an electronic scaling device.
The sensing unit may be a Geiger-Müller tube, a proportional counter, a scintillation
detector in which a photomultiplier tube is employed in conjunction with a scintillator, or
a solid-state semi-conductor.
Geiger-Müller counters and proportional counters are generally used for the measurement
of the beta emitters. Scintillation counters employing liquid or solid phosphors may be
used for the measurement of alpha, beta, and gamma emitters. Solid-state devices may
also be used for alpha, beta, and gamma measurements. The electronic circuitry
associated with a detector system usually consists of a high-voltage supply, an amplifier,
a pulse-height selector, and a scaler, a rate meter, or other readout device. When the
electronic scaling device or the scaler in a counting assembly is replaced by an electronic
integrating device, the resultant assembly is a rate meter. Rate meters are used for the
purpose of monitoring and surveying radioactivity and are somewhat less precise as
measuring instruments than the counters. Ionization chambers are often used for
measuring gamma-ray emitters and, similar type of thin-walled instruments for measuring
X-rays. Dose calibrators are ionization chambers used for measuring the amount of
radioactivity in a vial or the dose to a patient in a syringe.
Radiation from a radioactive source is emitted in all directions this is, isotropically.
Procedures for the standardization and measurement of such sources by means of a count
of the emissions in all directions are known as 4π-counting; those based on a count of the
emissions in a solid angle of 2π steradians are known as 2π-counting; and those based on
a fraction of the emissions defined by the solid angle subtended from the detector to the
source are known as counting in a fixed geometry. It is customary to assay the
radioactivity of a preparation by comparison with a standardized preparation using
identical geometry conditions. The validity of such an assay is critically dependent upon
the reproducibility of the spatial relationships of the source to the detector and its
surroundings and upon the accuracy of the standardized preparation. In the primary
standardization of radionuclides coincidence techniques are employed in preference to
simple 4π-counting whenever the decay scheme of the radionuclide permits. One of the
most commonly employed coincidence techniques is 4π-beta/gamma coincidence
Working document QAS/07.242
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counting, which is used for nuclides in which some or all of the disintegrations are
followed by prompt photon emission. An additional adjacent detector, sensitive only to
photons, is used to measure the efficiency in the 4π-counter of those disintegrations with
which the photons are coincident. 4π-Gamma/gamma coincidence counting techniques
are often employed for the standardization of pure gamma emitters.
The construction and performance of instruments and accessory apparatus could vary to a
great extent. The preparation of samples must therefore, be modified to obtain
satisfactory results with a particular instrument. The operator must carefully follow the
manufacturer's instructions for obtaining optimum instrument performance. The results
must be substantiated by careful examination of known samples. Proper instrument
functioning and reliability must be monitored on a day-to-day basis through the use of
secondary reference preparations.
Radioactivity occurring in materials of construction, or caused by cosmic rays, and to
spontaneous discharges in the atmosphere contributes to what is known as the
background activity. All sample radioactivity measurements must be corrected by
subtracting the respective background activity.
When counting of samples at high activity levels, corrections must be made also for loss
of counts due to inability of the equipment to resolve pulses arriving in close succession.
Such coincidence-loss corrections must be made prior to the background correction.
The corrected count rate, R, is given by the formula:
Where r is the observed count rate, and τ is the resolving time.
A radioactivity count is a statistical value, i.e., it is a measure of nuclear decay
probabilities, and is not exactly constant over any given time interval. The magnitude of
the standard deviation is approximately equal to the square root of the number of counts.
In general, at least 10 000 counts are necessary to obtain a standard deviation of 1 %.
Absorption
Ionizing radiation is absorbed in the material surrounding the source of the radiation.
Such absorption occurs in air, in the sample itself (self-absorption), in sample coverings,
in the window of the detection device, and in any special absorbers placed between the
sample and the detector. Since alpha particles have a short range of penetration in matter,
beta particles have a somewhat greater range, and gamma rays are deeply penetrating,
identification of the type and energy of radiation emitted from a particular radionuclide
may be determined by the use of absorbers of varying thickness. In practice, this method
is seldom used, and that too mainly in connexion with beta emitters. Therefore, variations
in counting rate due to (small) differences in thickness and density of sample containers
could give rise to major problem with beta emitters and with X-ray emitters, such as
Working document QAS/07.242
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iodine-125. Plastic containers, in which variations of density and thickness are minimal,
are therefore often employed in such cases. Plastic tubes with defined density and
thickness are therefore employed frequently.
The absorption coefficient (µ), which
mg/cm2, or the half-thickness (the
radioactivity by a factor of two), is
radiation emitted by a radionuclide.
radiation.
is the reciprocal of the
thickness of absorber
commonly determined
This equation is valid
“thickness” expressed in
required to reduce the
to characterize the beta
only for monoenergetic
Radiation spectrometry
Crystal scintillation spectrometry
When the energy of beta or gamma radiation is dissipated within some materials known
as scintillators, light is produced in an amount proportional to the energy dissipated. This
quantity of light may be measured by suitable means, and is proportional to the energy
absorbed in the scintillator. The light emitted under the impact of a gamma photon or a
beta particle is converted into an electric output pulse by a photomultiplier. Scanning of
the output pulses with a suitable pulse-height analyser results in an energy spectrum of
the source.
The scintillators most commonly used for gamma spectrometry are single crystals of
thallium-activated sodium iodide. Gamma-ray scintillation spectra show one or more
sharp, characteristic photoelectric peaks, corresponding to the energies of the gamma
radiation of the source. They are thus useful for identification purposes and also for the
detection of gamma-emitting impurities in a preparation. These peaks are accompanied
by other peaks due to secondary effects of radiation on the scintillator and its
surroundings, such as backscatter, positron annihilation, coincidence summing, and
fluorescent X-rays. In addition, broad bands known as the Compton continua arise from
the scattering of the gamma photons in the scintillator and in surrounding materials.
Calibration of the instrument is performed with the use of known samples of
radionuclides whose energy spectra have been characterized. The shape of the spectrum
produced will vary with the instrument used, owing to such factors as differences in the
shape and size of the crystal, in the shielding materials used, the distance between the
source and the detector, and in the types of discriminator employed in the pulse-height
analysers. When using the spectrum for identification of radionuclides it is therefore
necessary to compare the spectrum with that of a known sample of the radionuclide
obtained in the same instrument under identical conditions.
Certain radionuclides, for example, iodine-125, emit characteristic X-rays of well-defined
energies that will produce photoelectric peaks in a suitable gamma spectrometer. Beta
radiation also interacts with the scintillators, but the spectra are continuous and diffuse
and generally of no use for identification of the radionuclide or for the detection of betaemitting impurities in a radiopharmaceutical preparation.
Working document QAS/07.242
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Semiconductor detector spectrometry
Gamma-ray spectra may be obtained using solid-state detectors. The peaks obtained do
not suffer to the same extent the broadening shown in crystal scintillation spectrometry,
and the resolution of gamma photons of similar energies is very much improved.
However, the efficiencies of such detectors are much lower.
The energy required to create an electron-hole pair or to promote an electron from the
valence band to the conduction band in a semi-conductor is far less than the energy
required to produce a photon in a scintillation crystal. In gamma-ray spectrometry a high
purity germanium (HPGe) detector can provide an energy resolution of 0.14-0.18 % for
the 1.33 MeV photon of cobalt-60.
Liquid scintillation counting
For beta-emitters like 35S, 14C and 3H, where self-absorption of the low-energy beta
particles is significant, the preferred counting method is by liquid scintillation, which can
occasionally be employed also for emitters of X-rays, alpha-particles, and gamma-rays. If
the sample to be counted is dissolved in, or mixed with, a solution of an appropriate
scintillator material, the decay energy from the sample is converted into light photons.
These are sensed by a photomultiplier, which converts them into an electric pulse, whose
intensity is proportional to the energy of the initial radiation. Thus, simultaneous counting
of several radionuclides differing in the energy of emitted radiation can be effected with
suitable discriminators (pulse-height analysers), provided the energy separation is
adequate. Detection efficiencies approaching 95 % for 14C and 60 % for 3H are reached
because self-absorption is minimized.
The scintillator (to check the chemical) solute usually consists of a polycyclic aromatic
compound, such as p-terphenyl or 2,5-diphenyloxazole (primary solute), together with a
secondary solute, such as 1,4-di[2-(4-methyl-5-phenyloxazole)]benzene (DimethylPOPOP), that shifts the wavelength of the light emitted to match the highest sensitivity of
the photomultiplier tube. Water-immiscible solvents, such as toluene, or water-miscible
solvents, such as dioxan, can be used. To facilitate the counting of aqueous solutions,
special solvents have been developed. Alternatively, samples may be counted as
suspensions in scintillator gels. As a means of attaining compatibility and miscibility with
aqueous specimens to be assayed, many additives, such as surfactants and solubilizing
agents, are also incorporated into the scintillator. For accurate determination of sample
radioactivity, care must be taken to prepare a sample that is truly homogeneous. The
presence of impurities and colour in the solution causes a decrease in the number and
energy of photons reaching the photomultiplier tube; such a decrease is known as
quenching. Accurate radioactivity measurement requires correcting for count-rate loss
due to quenching. Solutions containing organic scintillators are prone to photo-excitation
and samples may need to be prepared in subdued light and kept in darkness before and
during counting process.
Half-life period
The time in which the radioactivity decreases to one-half its original value.
Working document QAS/07.242
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EXPLANATORY NOTE. The rate of radioactive decay is constant and characteristic
for each individual radionuclide. The exponential decay curve is described
mathematically by the equation:
where N is the number of atoms at elapsed time t, No is the number of atoms when t = 0,
and λ is the disintegration constant characteristic of each individual radionuclide. The
half-life period is related to the disintegration constant by the equation:
Radioactive decay corrections are calculated from the exponential equation, or from
decay tables, or are obtained from a decay curve plotted for the particular radionuclide
involved (see Fig. 1).
FIG. 1. MASTER DECAY CHART
Working document QAS/07.242
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Physical half-life
The physical half-life of a radionuclide (T½ p) is the time in which the amount of
radioactivity decreases to one half of its original value. Although the time of decay of an
individual atom can not be determined, large numbers of atoms will obey statistical
considerations and calculations of activity versus time can be carried out. The rate of
decay for a collection of atoms (N) of the same radionuclide is constant and characteristic
for each individual radionuclide. The exponential decay is described by the equation:
N = N O e − λt
Where N is the number of atoms after an elapsed time t. N0 is the number of atoms at
time t = 0 and λ is the decay constant characteristic for a given nuclide. This relationship
is commonly referred to as the decay law equation. Where the activity of a quantity of
radioactive substance is known at a certain time its activity at any other time can be
determined by using the decay law relationship. The physical half-life is related to the
decay constant by the equation:
T1/ 2 =
0.693
λ
In addition to the use of the decay law formula radioactivity can be determined at
different times using decay tables or decay curves plotted for the specific radionuclide.
Biological half-life
The biological half-life (T½b) of a radiopharmaceutical is the time taken for the
concentration of the pharmaceutical to be reduced 50% of its maximum concentration in
a given tissue, organ or whole body, not considering radioactive decay.
Effective half-life
The effective half-life (T½e) is the actual half-life of a radiopharmaceutical in a given
tissue, organ or whole body and is determined by a relationship including both the
physical half life and biological half-lives. The effective half-life is important in
calculation of the optimal dose of radiopharmaceutical to be administered and in
monitoring the amount of radiation exposure. It can be calculated from the formula:
T1 / 2 e =
T1 / 2 p xT1 / 2b
T1 / 2 p + T1 / 2b
Where T1/2p and T1/2b are the physical and biological half-lives respectively.
Physical characteristics of clinically relevant radionuclides.
For detailed information on physical characteristics including parent half life, daughter
half life, decay mode, energy, end-point energy intensity, dose and daughter nucleus refer
to IAEA nuclear data base http://www-nds.iaea.org/nudat/radform.html or
www.nchps.org http://www.nndc.bnl.gov/nudat2/decaysearchdirect
Working document QAS/07.242
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Key considers include, parent nuclide, parent half life, decay mode (Beta +/- ; gamma and
x-ray radiations, electrons), energy (keV), end-point energy intensity, dose (Mev/Bq-1)
See attached list of key radionuclide used in Nuclear medicine
Radionuclidic purity
The radionuclidic purity of a preparation is that percentage of the total radioactivity that
is present in the form of the stated radionuclide.
EXPLANATORY NOTE. Some radionuclides decay into nuclides that are themselves
radioactive: these are referred to as mother (or parent) and daughter radionuclides
respectively. Such daughter radionuclides are often excluded when calculating the
radionuclidic purity; for example, iodine-131 will always contain its daughter xenon-131
m, but this would not be considered an impurity because its presence is unavoidable.
In employing the definition, the radioactivity must be measured in appropriate units: that
is, in the number of nuclear transformations that occur in unit time (in terms Becquerels).
If, for example, a preparation stated to be iodine-125 is known to contain 99MBq of
iodine-125 and 1MBq of iodine-126, and no other radionuclide, then the preparation is
said to be of 99% radionuclidic purity. It will be noted that the relative amounts of iodine125 and iodine-126, and hence the radionuclidic purity, will change with time. An
expression of radionuclidic purity must therefore contain a statement of the time, such as:
“Not more than 1% of the total radioactivity is due to iodine-126 at the reference date
stated on the label”.
It is clear that, in order to give a statement of the radionuclidic purity of a preparation, the
activities (and hence the identities) of every radionuclide present must be known. There
are no simple and certain means of identifying and measuring all the radionuclidic
impurities that might be present in a preparation. An expression of radionuclidic purity
must either depend upon the judgement of the person concerned, or it must be qualified
by reference to the method employed, for example: “No radionuclidic impurities were
detected by gamma scintillation spectrometry using a sodium iodide detector.”
Requirements for radionuclide purity
Requirements for radionuclide purity are specified in two ways:
1. By expression of a minimum level of radionuclide purity. Unless otherwise stated
in the individual monograph, the gamma-ray spectrum, should not be significantly
different from that of a standardized solution of the radionuclide before the expiry
date is reached.
2. By expression of maximum levels of specific radionuclide impurities in the
individual monographs. In general, such impurities are those that are known to be
likely to arise during the production of the material – for example, thallium-202
(t1/2=12.23d) in the preparation of thallium-201 (t1/2 =73.5h).
Working document QAS/07.242
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It is evident that while the above requirements are necessary, they are not in themselves
sufficient to ensure that the radionuclide purity of a preparation is sufficient for human
use. The manufacturer is responsible to ensure the quality of his products, and especially
to examine preparations of short-lived radionuclides for long-lived impurities after a
suitable period of decay. In this way, the manufacturer ensures that the manufacturing
processes (refer to WHO GMP guidance) employed are producing materials of
appropriate quality. In particular, the radionuclide composition of certain preparations is
determined by the chemical and isotopic composition of the target material, which is
irradiated with neutrons or charged particles, and trial preparations are advisable when
new batches of target material are employed.
Radioactive concentration
The radioactive concentration of a solution refers to the amount radioactivity per unit
volume of the solution. As with all statements involving radioactivity, it is necessary to
include a reference date and time of standardization. For radionuclides with a half-life
period of less than one day, a more precise statement of the reference time is required.
In addition, the term radioactive concentration is generally applied to solutions of a
radioactive solute. The radioactive concentration of a solution refers to the amount of
radioactivity per unit volume of the solution. An example of units for radioactive
concentration would be megaBecquerels per millilitre (MBq/ml). Since the radioactive
concentration will change with time due to decrease in the nuclide radioactivity it is
always necessary to provide a reference time. For short lived radionuclides the reference
time will be more precise including time of day in addition to date.
Specific radioactivity (or specific activity)
The specific activity of a preparation of a radioactive material is the radioactivity per unit
mass of the element or of the compound concerned.
The specific activity of a given radioisotope refers to the disintegration rate per unit mass
of the element. For example, a fresh solution of 99mTc will have a specific activity of:
As = NxA
Where As=specific activity, and N=the number of 99mTc atoms in one gram of pure
technetium. N is calculated as:
N=6.023 x 1023 (atoms/mole)/(99 grams/mole)
However, the following note must be taken into consideration to calculate the specific
activity of a formulated compound.
EXPLANATORY NOTE. It is usual to specify the radionuclide concerned and also it is
necessary to express the time thus: “100MBq of iodine-131 per mg of MIBG at 12.00
hours GMT on 1 January 2006”.
Specific radioactivity is often not determined directly but is calculated from knowledge
of the radioactive concentration of the solution and of the chemical concentration of the
radioactive compound. Thus, if a solution contains x MBq of 131I per ml, and if the 131I is
Working document QAS/07.242
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entirely in the chemical form of MIBG of which the concentration is y mg per ml, then at
that time the specific activity is:
x/y mCi of iodine-131 per mg of MIBG.
Where necessary, the radiochemical purity of the preparation (see below) must be taken
into account.
The term employed in radiochemical work is “specific activity”. As the word, “activity”
has other connotations in a pharmacopoeia, the term should, where necessary, be
modified to “specific radioactivity” to avoid ambiguity.
Radiochemical purity
The radiochemical purity of a preparation is that percentage of the stated radionuclide
that is present in the stated chemical form. As radiochemical purity may change with time,
mainly because of radiation decomposition, the result of the radiochemical purity test
should be started at given date and if necessary hour indicating when the test was carried
out. The radiochemical purity limit should be valid during the whole shelf-life.
EXPLANATORY NOTE. If, for example, a preparation of 99mTc-DTPA is stated to be 99
% radiochemically pure, then 99% of the technetium-99m is present in the form of DTPA
(diethylenetriamine-pentaacetic acid) complex. Radiochemical impurities might include
such substances as reduced-hydrolyzed 99mTc or free 99mTc-pertechnetate anion
The possible presence of radionuclide impurities is not taken into account in the
definition. If the radionuclide impurity is not isotopic with the stated radionuclide, then it
cannot possibly be in the identical chemical form. If the radionuclide impurity is isotopic
with the stated radionuclide, it could be, and indeed is likely to be, in the same chemical
form.
Radiochemical impurities may arise during the preparation of the material or during
storage, because of ordinary chemical decomposition or, what is often more important,
because of radiation decomposition (that is, because of the physical and chemical effects
of the radiation-radiolysis).
Starting material
In general for radiopharmaceutical industry these are normally produced on a small scale
and supplied by specialized producer or laboratories. However starting materials for use
as radiopharmaceuticals starting material must meet all of the quality criteria suitable for
the intended use. Principles for starting material stated in WHO/Pharm/98.605 report
should be followed.
Starting materials designated to be Ph. Int. quality should meet the respective
requirements before the material can be labelled and accepted for use. The moment
starting material is designated for pharmaceutical purposes, it should be appropriately
controlled during manufacture, handling and distribution. Proper identification of starting
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materials is indispensable under all circumstances, even when it is radioactive and
accompanied by a reliable certificate of analysis. Proper identification of starting
materials is indispensable under all circumstances.
The manufacturing facilities and the manufacturing process for a substance or dosage
form that is the subject of a monograph in the Ph. Int. must meet the current WHO
requirements of Good Manufacturing Practice 1 . Statements under the heading
"manufacture" draw attention to particular aspects of the manufacturing process but are
not necessarily comprehensive. They may be in the form of mandatory instructions to
manufacturers or, where clear from the form of wording used, they may provide
guidance. In the general monographs for dosage forms, information is given that is
intended to provide broad guidelines concerning the main steps to be followed during
production, indicating those that are most important. For the current recommendations,
consult the WHO Medicines web site (http://www.who.int/medicines) or IAEA web site.
Where radiopharmaceuticals contain serum albumin these substances must be
manufactured in accordance with the WHO "Recommendations on Risk of Transmitting
Animal Spongiform Encephalopathy Agents via Medicinal Products" reproduced in the
section Supplementary Information.
Carriers
The mass of radioactive material usually encountered in radioactive pharmaceuticals is
often too small to be measured by ordinary chemical or physical methods. Since such
small amounts may not be subject to the usual methods of separation and purification, a
carrier, in the form of inactive material, either isotopic with the radionuclide, or nonisotopic, but chemically similar to the radionuclide, may be added during processing and
dispensing to permit ready handling. Thus sodium phosphate carrier is present in “Natrii
Phosphatis (32P) Injectio” and rhenium is used as a carrier in certain colloidal
preparations of technetium-99m. The amount of carrier added must be sufficiently small
for it not to cause undesirable physiological effects. The mass of an element formed in a
nuclear reaction may be exceeded by that of the inactive isotope present in the target
material or in the reagents used in the separation procedures.
Carrier-free
Radioactive preparations in which no carrier is intentionally added during the
manufacture or processing are often loosely referred to as carrier-free. The designation
no-carrier-added is sometimes used to indicate that no dilution of the specific activity has
taken place by design although carrier may exist due to the natural presence of nonradioactive element or compound accumulated during the production of the radionuclide
or preparation of the compound in question. In some situations it will be necessary to add
carrier to enhance chemical, physical or biological properties of the radiopharmaceutical.
Carrier-free specific activity (SA) can be determined by a consideration of the
1
WHO good manufacturing practices: main principles for pharmaceutical products. In: Quality assurance
of pharmaceuticals. A compendium of guidelines and related materials. Good manufacturing practices and
inspection. Volume 2, Second updated edition. Geneva, World Health Organization, 2007.
Working document QAS/07.242
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relationship between activity A, the number of radioactive atoms present N and the decay
constant λ where λ = 0.693/T1/2.
⎛ 0.693 ⎞
⎟⎟
A = Nλ = N ⎜⎜
⎝ T1 / 2 ⎠
The specific activity of radioactive materials that are not carrier-free can be determined
by measuring both the radioactivity and the physical amount of material present within
the element or compound of interest. Accurate determination, where a material has a high
specific activity, may be difficult due to limitations in obtaining an accurate
determination of the amount of the substance present by standard physical or chemical
analysis.
Excipient
As with starting materials appropriate control of excipients during manufacture, handling
and distribution is important. The actual quantity of radioactive material compared with
quantities of excipients is normally very small therefore excipients can greatly influence
quality of RP.
Compounding
Compounded radiopharmaceuticals are not for sale and are not to be advertised.
Compounding includes formulation of radiopharmaceutical reagent kits from raw
ingredients
for
radiopharmaceuticals
preparation,
adding
reagents
to
approved/unapproved commercial kits to modify or enhance performance of
radiopharmaceuticals (shelf life extension, fractionation) and/or synthesis from raw
materials. Compounding should follow recognized pharmacopoeial protocols whenever
available; approval by institutional committee is otherwise required. The process of
compounding radiopharmaceuticals must be under the supervision and responsibility of
recognized nuclear physician or suitably qualified professional, ideally a radiopharmacist.
Within the radiopharmaceutical industry the range of associated risk of product failure
varies from manufacturing, compounding and dispensing. RP designated to be Ph. Int.
quality should meet the respective requirements before the material can be labelled and
accepted for use. Compounding is limited to clinical practice according to medical
doctor’s prescription or requisition for a specific patient. Patent-protected
radiopharmaceuticals should not be compounded. When, however, patented reagent kits
cannot be readily obtainable from a commercial source, limited compounding shall be
done to meet the urgent medical needs of an identified individual patient; in this case the
prescriber shall be informed that a reagent kit will be compounded to replace the
commercial product.
Dispensing
Dispensing of radiopharmaceutical is distinct from compounding in that a
radiopharmaceutical is prepared with the use of a commercially approved/authorized
reagent kit (“cold kit”) by reconstituted using another commercially approved/authorized
pharmaceutical, radioactive material, even an elute from commercially
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approved/authorized generator system. All aspects are undertaken in accordance with the
commercially approved/authorized manufacturer and supplier.
Critical Organ
The Critical Organ is the organ or tissue which receives the highest radiation dose. This
may not be the target tissue and therefore the dose to the critical organ will determine the
maximum safe dose which can be administered. This is primarily of importance with
respect to therapeutic radiopharmaceuticals.
End-user assessment
Simple, non-destructive tests are advised which adequately identify radiopharmaceutical.
If non-licensed or non-approved/registered radiopharmaceuticals under national rules are
used, a detailed certificate of analysis or certificate of compliance is essential. In addition,
the essential tests which give sufficient assurance of quality must be undertaken to allow
safe use in patients.
67
For example, for Gallium Citrate ( Ga) injection: Quick end user check alternative
method: 6 cm ITLC strip, drop at 1 cm from lower end, develop in methanol-acetic acid
(9:1) mixture, cut at 3 cm from origin. 67Ga citrate remains at the origin.
***