Cyanocobalamin (58Co) solution
EUROPEAN PHARMACOPOEIA 5.0
01/2005:0270 — as mobile phase at a flow rate of 1.0 ml/min a mixture
prepared as follows : mix 26.5 volumes of methanol R and
58
73.5 volumes of a 10 g/l solution of disodium hydrogen
CYANOCOBALAMIN ( Co) SOLUTION
phosphate R adjusted to pH 3.5 using phosphoric acid R
and use within 2 days,
Cyanocobalamini (58Co) solutio
— a radioactivity detector adjusted for cobalt-58,
DEFINITION
— as detector a spectrophotometer set at 361 nm,
Cyanocobalamin (58Co) solution is a solution of
— a loop injector.
[58Co]-α-(5,6-dimethylbenzimidazol-1-yl)cobamide cyanide
Inject 100 µl of the solution to be examined and record
and may contain a stabiliser and an antimicrobial
the chromatogram for three times the retention time of
preservative. Cobalt-58 is a radioactive isotope of cobalt
cyanocobalamin. Determine the peak areas and calculate
and may be produced by neutron irradiation of nickel.
the percentage of cobalt-58 present as cyanocobalamin.
58
Cyanocobalamin ( Co) may be prepared by the growth of
Inject 100 µl of the reference solution and record the
58
suitable micro-organisms on a medium containing ( Co)
chromatogram for 30 min.
cobaltous ion. The solution contains not less than 90.0 per
cent and not more than 110.0 per cent of the declared
RADIOACTIVITY
cobalt-58 radioactivity at the date stated on the label. Not
Measure the radioactivity using suitable counting equipment
less than 90 per cent of the cobalt-58 is in the form of
by comparison with a standardised cobalt-58 solution or by
cyanocobalamin.
measurement in an instrument calibrated with the aid of
such a solution.
CHARACTERS
A clear, colourless or slightly pink solution.
STORAGE
Cobalt-58 has a half-life of 70.8 days and emits beta (β+)
Store protected from light at a temperature of 2 °C to 8 °C.
radiation and gamma radiation.
IDENTIFICATION
A. Record the gamma-ray spectrum using a suitable
instrument. The spectrum does not differ significantly
from that of a standardised cobalt-58 solution.
Standardised cobalt-58, cobalt-57 and cobalt-60 solutions
are available from laboratories recognised by the
competent authority. The most prominent gamma
photons of cobalt-58 have energies of 0.511 MeV
(annihilation radiation) and 0.811 MeV.
B. Examine the chromatograms obtained in the test
for radiochemical purity. The principal peak in the
radiochromatogram obtained with the solution to be
examined has a retention time similar to that of the
peak in the chromatogram obtained with the reference
solution.
TESTS
pH (2.2.3). The pH of the solution is 4.0 to 6.0.
RADIONUCLIDIC PURITY
Record the gamma-ray spectrum using a suitable instrument
having adequate resolution and calibrated with the aid of
standardised cobalt-58, cobalt-57 and cobalt-60 solutions.
The spectrum does not differ significantly from that of the
standardised cobalt-58 solution. Determine the relative
amounts of cobalt-58, cobalt-57 and cobalt-60 present.
Cobalt-57 has a half-life of 271 days and its presence is shown
by gamma photons of energy 0.122 MeV. Cobalt-60 has a
half-life of 5.27 years and its presence is shown by gamma
photons of energies 1.173 MeV and 1.332 MeV. Not more
than 1 per cent of the total radioactivity is due to cobalt-60
and not more than 2 per cent of the total radioactivity is due
to cobalt-57, cobalt-60 and other radionuclidic impurities.
RADIOCHEMICAL PURITY
Examine by liquid chromatography (2.2.29).
Reference solution. Dissolve 10 mg of cyanocobalamin CRS
in the mobile phase and dilute to 100 ml with the mobile
phase. Dilute 2 ml of the solution to 100 ml with the mobile
phase. Use within 1 h.
The chromatographic procedure may be carried out using :
— a stainless steel column 0.25 m long and 4 mm in
internal diameter packed with octylsilyl silica gel for
chromatography R (5 µm),
822
01/2005:1325
FLUDEOXYGLUCOSE (18F) INJECTION
Fludeoxyglucosi (18F) solutio iniectabilis
DEFINITION
Fludeoxyglucose (18F) injection is a sterile
solution of 2-[18F]fluoro-2-deoxy-D-glucopyranose
(2-[18F]fluoro-2-deoxy-D-glucose) for diagnostic use. The
injection contains not less than 90.0 per cent and not
more than 110.0 per cent of the declared fluorine-18
radioactivity at the date and time stated on the label. Not
less than 95 per cent of the radioactivity corresponds to
fluorine-18 in the form of 2-[18F]fluoro-2-deoxy-D-glucose
and 2-[18F]fluoro-2-deoxy-D-mannose, with the
2-[18F]fluoro-2-deoxy-D-mannose fraction not exceeding
10 per cent of the total radioactivity. Not less than 99.0 per
cent of the radioactivity corresponds to fluorine-18. The
content of 2-fluoro-2-deoxy-D-glucose is not more than 10 mg
per maximum recommended dose of injection.
PRODUCTION
RADIONUCLIDE PRODUCTION
Fluorine-18 is a radioactive isotope of fluorine which may be
produced by various nuclear reactions induced by proton
irradiation of oxygen-18, deuteron irradiation of neon-20,
helium-3 or helium-4 irradiation of oxygen-16.
RADIOCHEMICAL SYNTHESIS
2-[18F]Fluoro-2-deoxy-D-glucose may be prepared by various
chemical synthetic pathways, which lead to different
products in terms of specific radioactivity, by-products and
possible impurities.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
Most widely used is the method of phase transfer catalysed
nucleophilic substitution of 1,3,4,6-tetra-O-acetyl-2-Otrifluoromethanesulphonyl-β-D-mannopyranose with
[18F]fluoride. Generally, [18F]fluoride is adsorbed on
an anion-exchange resin and eluted with a solution
of potassium carbonate which is then evaporated to
dryness. Addition of a phase transfer catalyst such as
an aminopolyether in dry acetonitrile may be used to
enhance the nucleophilicity of the [18F]fluoride so that
it reacts easily with the tetra-acetylated mannosyltriflate
at elevated temperature. Hydrolysis under either alkaline
or acidic conditions yields 2-[18F]fluoro-2-deoxy-D-glucose.
Hydrolysis using hydrochloric acid may lead to the
formation of 2-chloro-2-deoxy-D-glucose. Hydrolysis
under alkaline conditions may lead to the formation of
2-[18F]fluoro-2-deoxy-D-mannose as a by-product.
Variations of the method substitute the aminopolyether by
a tetra-alkyl ammonium salt, or use solid phase catalysed
nucleophilic substitution on derivatised anion-exchange
resin, e.g. derivatised with 4-(4-methylpiperidino)pyridine.
Electrophilic pathways for production of 2-[18F]fluoro-2-deoxyD-glucose proceed by the reaction of molecular [18F]fluorine
or [18F]acetylhypofluorite with 3,4,6-tri-O-acetyl-D-glucal.
[18F]Acetylhypofluorite is obtained by conversion of
molecular [18F]fluorine on a solid complex of acetic acid and
potassium acetate. The production of molecular [18F]fluorine
requires the addition of small amounts of fluorine to the neon
target gas, usually from 0.1 per cent to 1 per cent, resulting in
the reduction of the specific radioactivity of the end-product.
Hydrolysis of the O-acetyl protected [18F]fluorinated sugar
yields 2-[18F]fluoro-2-deoxy-D-glucose and usually small
amounts of 2-[18F]fluoro-2-deoxy-D-mannose.
The preparation can be purified by serial chromatography
on combinations of ion-retardation resin, ion-exchange resin,
alumina and octadecyl derivatised silica gel. Removal of the
phase transfer catalyst can be achieved by different methods,
all using combinations of separation cartridges.
Production systems and their performance comply with the
requirements set by the competent authority.
STARTING MATERIALS
1. Target materials
Each batch of target material must be tested in special
production runs before its use in routine fluorine-18
production and manufacture of the preparation, to ensure
that under specified conditions, the target yields fluorine-18
in the desired quantity and quality.
2. Precursors for organic synthesis
It is recommended to test the precursors in production runs
before their use for the manufacture of the preparation,
to ensure that under specified production conditions, the
precursors yield the preparation in the desired quantity and
quality.
1,3,4,6-Tetra-O-acetyl-2-O-trifluoromethanesulphonylβ-D-mannopyranose. Examine by infrared absorption
spectrophotometry (2.2.24), comparing with the Ph. Eur.
reference spectrum of 1,3,4,6-tetra-O-acetyl-2-Otrifluoromethanesulphonyl-β-D-mannopyranose.
Melting point (2.2.14) : 119 °C to 122 °C.
3,4,6-Tri-O-acetyl-D-glucal. Examine by infrared absorption
spectrophotometry (2.2.24), comparing with the Ph. Eur.
reference spectrum of 3,4,6-tri-O-acetyl-D-glucal.
Melting point (2.2.14) : 53 °C to 55 °C.
CHARACTERS
A clear, colourless or slightly yellow solution.
General Notices (1) apply to all monographs and other texts
Fludeoxyglucose (18F) injection
Fluorine-18 has a half-life of 109.8 min and emits positrons
with a maximum energy of 0.633 MeV, followed by
annihilation gamma radiation of 0.511 MeV.
IDENTIFICATION
A. Record the gamma-ray spectrum using a suitable
instrument. The only gamma photons have an energy
of 0.511 MeV ; and depending on the measurement
geometry, a sum peak of 1.022 MeV may be observed.
B. It complies with the test for radionuclidic purity (see
Tests).
C. Examine the chromatograms obtained in test (a)
for radiochemical purity. The principal peak in the
radiochromatogram obtained with the test solution has
approximately the same retention time as the principal
peak in the chromatogram obtained with reference
solution (b).
TESTS
pH (2.2.3). The pH of the injection is 4.5 to 8.5.
Sterility. It complies with the test for sterility
prescribed in the monograph on Radiopharmaceutical
preparations (0125). The injection may be released for use
before completion of the test.
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V
being the maximum recommended dose in millilitres. The
injection may be released for use before completion of the
test.
CHEMICAL PURITY
Particular tests for chemical purity may be omitted if the
substances mentioned are not used or cannot be formed in
the production process
(a) 2-Fluoro-2-deoxy-D-glucose and 2-chloro-2-deoxy-Dglucose. Examine by liquid chromatography (2.2.29).
Test solution. The preparation to be examined.
Reference solution (a). Dissolve 10 mg of glucose R in
water R and dilute to 100 ml with the same solvent.
Reference solution (b). Dissolve 10 mg of
2-fluoro-2-deoxy-D-glucose R in water R and dilute
to V with the same solvent, V being the maximum
recommended dose in millilitres.
Reference solution (c). Dissolve 1.0 mg of
2-chloro-2-deoxy-D-glucose R in water R and dilute
to 2.0 ml with the same solvent. Dilute 1 ml of this
solution to V with the same solvent, V being the maximum
recommended dose in millilitres.
The chromatographic procedure may be carried out using :
— a column 0.25 m long and 4.0 mm in internal diameter
packed with strongly basic anion-exchange resin for
chromatography R (10 µm),
— as mobile phase at a flow rate of 1 ml/min 0.1 M sodium
hydroxide protected against contamination by carbon
dioxide,
— a suitable radioactivity detector for radiochemical purity
testing,
— a detector suitable for carbohydrates in the required
concentration range,
— a loop injector,
maintaining the column at a constant temperature between
20 °C and 30 °C.
Equilibrate the column with the mobile phase until a stable
baseline is achieved.
Inject separately reference solutions (a), (b) and (c).
If the validation studies exclude the formation of
2-chloro-2-deoxy-D-glucose inject separately reference
823
Fludeoxyglucose (18F) injection
EUROPEAN PHARMACOPOEIA 5.0
solutions (a) and (b). Continue the chromatography for twice
the retention time of D-glucose, 2-fluoro-2-deoxy-D-glucose
and when required, 2-chloro-2-deoxy-D-glucose respectively.
Inject the test solution. The chromatogram obtained
with the detector for carbohydrates shows a principal
peak corresponding to D-glucose (test solutions from
nucleophilic pathways) or 2-fluoro-2-deoxy-D-glucose
(test solutions from electrophilic pathways). When
the chromatograms are recorded in the prescribed
conditions, 2-chloro-2-deoxy-D-glucose elutes after
2-fluoro-2-deoxy-D-glucose, but their corresponding peaks
may not be completely resolved. In the chromatogram
obtained with the test solution, the areas of the
peaks corresponding to 2-fluoro-2-deoxy-D-glucose and
2-chloro-2-deoxy-D-glucose are not greater than the
areas of the peaks in the chromatograms obtained with
reference solution (b) and/or reference solution (c)
(10 mg of 2-fluoro-2-deoxy-D-glucose per V and 0.5 mg of
2-chloro-2-deoxy-D-glucose per V respectively).
(b) Aminopolyether. This test is performed only on the
bulk solution before addition of sodium chloride by the
producer and it is not intended for the final preparation to
be injected. Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel plate R.
Test solution. The preparation to be examined.
Reference solution. Dissolve 0.110 g of aminopolyether R in
water R and dilute to 10.0 ml with the same solvent. Dilute
0.2 ml of this solution to V with the same solvent, V being
the maximum recommended dose in millilitres.
Apply separately to the plate 2 µl of the test solution and 2 µl
of the reference solution. Develop over a path of about 8 cm
using a mixture of 1 volume of ammonia R and 9 volumes
of methanol R. Allow the plate to dry in air for 15 min.
Expose the plate to iodine vapour for at least 10 min. In
the chromatogram obtained with the test solution the spot
corresponding to aminopolyether is not more intense than
the spot in the chromatogram obtained with the reference
solution (2.2 mg per V).
(c) Tetra-alkyl ammonium salts. Examine by liquid
chromatography (2.2.29).
Test solution. The preparation to be examined.
Reference solution. Dilute 2.1 ml of 0.1 M
tetrabutylammonium hydroxide to 20 ml with
water R. Dilute 1 ml of this solution to V with the same
solvent, V being the maximum recommended dose in
millilitres.
The chromatographic procedure may be carried out using :
— a column 0.125 m long and 4.0 mm in internal
diameter packed with octadecylsilyl silica gel for
chromatography R (5 µm),
— as mobile phase at a flow rate of 0.6 ml/min a mixture of
25 volumes of a 0.95 g/l solution of toluenesulphonic
acid R and 75 volumes of acetonitrile R,
— as detector a spectrophotometer set at 254 nm,
— a loop injector,
maintaining the column at a constant temperature between
20 °C and 30 °C.
Equilibrate the column with the mobile phase until a stable
baseline is obtained.
Inject the reference solution. Continue the chromatography
for twice the retention time of tetrabutylammonium ions.
Inject the test solution. In the chromatogram obtained with
the test solution, the area of the peak corresponding to
tetrabutylammonium ions is not greater than the area of
the peak in the chromatogram obtained with the reference
solution (2.75 mg per V).
824
(d) Solid phase derivatisation agent 4-(4-methylpiperidino)pyridine. Examine by ultraviolet
spectrophotometry (2.2.25).
Test solution. The preparation to be examined.
Reference solution. Dissolve 20 mg of 4-(4methylpiperidino)pyridine R in water R and dilute
to 100.0 ml with the same solvent. Dilute 0.1 ml of this
solution to V with the same solvent, V being the maximum
recommended dose in millilitres.
Measure the absorbance of the test solution and the reference
solution at the maximum of 263 nm. The absorbance of
the test solution is not greater than that of the reference
solution (0.02 mg per V).
(e) Residual solvents (2.4.24). The concentration of
acetonitrile does not exceed 4.1 mg per V, V being the
maximum recommended dose in millilitres. The injection
may be released for use before completion of the test.
RADIONUCLIDIC PURITY
Record the gamma-ray spectrum using a suitable instrument.
The half-life is between 105 min and 115 min. The injection
may be released for use before completion of the test.
RADIOCHEMICAL PURITY
A. Examine by liquid chromatography (2.2.29) as described
in test (a) for chemical purity.
When the chromatograms obtained with the radioactivity
detector are recorded in the prescribed conditions,
the principal peak in the chromatogram obtained
with the test solution has the same retention time as
the peak obtained with reference solution (b) using
the carbohydrate detector. The retention times of
2-[18F]fluoro-2-deoxy-D-mannose and [18F]fluoride are
approximately 90 per cent and approximately 50 per cent
of that of 2-[18F]fluoro-2-deoxy-D-glucose respectively.
Other peaks in the chromatogram may be due to partially
acetylated 2-[18F]fluoro-2-deoxy-D-glucose derivatives.
Calculate the percentage content of [18F]fluorinated
substances from the areas of the peaks in the
chromatogram obtained with the test solution.
The sum of the percentages of radioactivity
corresponding to 2-[18F]fluoro-2-deoxy-D-glucose
and 2-[18F]fluoro-2-deoxy-D-mannose is not less
than 95 per cent of the total radioactivity with the
2-[18F]fluoro-2-deoxy-D-mannose fraction not exceeding
10 per cent of the total radioactivity.
The method may underestimate or miss unhydrolysed or
partially hydrolysed 2-[18F]fluoro-2-deoxytetra-acetyl-Dglucose, since these intermediate reaction products may
further hydrolyse to the desired end-product under the
chromatographic conditions.
B. Examine by thin-layer chromatography (2.2.27) using a
TLC silica gel plate R.
Test solution. The preparation to be examined.
Apply 2 µl to 10 µl to the plate. Develop over a path
of 8 cm using a mixture of 5 volumes of water R and
95 volumes of acetonitrile R. Allow the plate to dry in air
for 15 min. Determine the distribution of radioactivity
using a suitable detector. Not less than 95 per cent of the
total radioactivity is found in the spot corresponding to
2-fluoro-2-deoxy-D-glucose (Rf about 0.45).
Possible contaminants are [18F]fluoride (Rf 0.0) ; partially
acetylated 2-[18F]fluoro-2-deoxy-D-glucose derivatives (Rf
about 0.8-0.95).
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
Flumazenil (N-[11C]methyl) injection
Synthesis of [5-methyl-11C]flumazenil
The most widely used method to obtain [5-methyl11
C]flumazenil is the N-alkylation of demethylflumazenil
with iodo[11C]methane in alkaline conditions in a solvent
such as dimethylformamide or acetone. The resulting
[5-methyl-11C]flumazenil can be purified by semi-preparative
liquid chromatography. For example, a column packed with
LABELLING
octadecylsilyl silica gel for chromatography eluted with a
The accompanying information specifies the particular
mixture of ethanol and water is suitable.
synthetic pathway of production. The label on the actual
PRECURSOR FOR SYNTHESIS
container states the maximum recommended dose in
Demethylflumazenil
millilitres.
Melting point (2.2.14) : 286 °C to 289 °C.
01/2005:1917 Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of
demethylflumazenil.
FLUMAZENIL (N-[11C]METHYL)
RADIOACTIVITY
Measure the radioactivity using suitable counting equipment
by comparison with a standardised fluorine-18 solution
or using an instrument calibrated with the aid of such a
solution. Standardised fluorine-18 solutions are available
from laboratories recognised by the competent authority.
INJECTION
Flumazenil (N-[11C]methyl)
solutio iniectabilis
DEFINITION
Sterile solution of ethyl 8-fluoro-5-[11C]methyl-6-oxo-5,6dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate
which may contain a stabiliser such as ascorbic acid.
Content : 90 per cent to 110 per cent of the declared
carbon-11 radioactivity at the date and time stated on the
label.
Content of flumazenil : maximum 50 µg in the maximum
recommended dose in millilitres.
CHARACTERS
Appearance : clear, colourless solution.
Half-life and nature of radiation of carbon-11 : see Table of
physical characteristics of radionuclides (5.7).
IDENTIFICATION
A. Gamma-ray spectrometry.
Results : the only gamma photons have an energy of
0.511 MeV and, depending on the measurement geometry,
a sum peak of 1.022 MeV may be observed.
B. It complies with test B for radionuclidic purity (see Tests).
C. Examine the chromatograms obtained in the test for
radiochemical purity.
Results : the principal peak in the radiochromatogram
obtained with the test solution is similar in retention time
to the principal peak in the chromatogram obtained with
reference solution (a).
TESTS
pH (2.2.3) : 6.0 to 8.0.
Sterility. It complies with the test for sterility
prescribed in the monograph on Radiopharmaceutical
preparations (0125). The injection may be released for use
PRODUCTION
before completion of the test.
RADIONUCLIDE PRODUCTION
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V
Carbon-11 is a radioactive isotope of carbon which is most
being the maximum recommended dose in millilitres. The
commonly produced by proton irradiation of nitrogen.
injection may be released for use before completion of the
Depending on the addition of either trace amounts of oxygen test.
or small amounts of hydrogen, the radioactivity is obtained
Flumazenil and impurity A. Liquid chromatography (2.2.29).
as [11C]carbon dioxide or [11C]methane, respectively.
Test solution. The preparation to be examined.
RADIOCHEMICAL SYNTHESIS
11
Reference solution (a). Dissolve 2.5 mg of flumazenil R in
[5-Methyl- C]flumazenil may be prepared by
5 ml of methanol R.
N-alkylation of ethyl 8-fluoro-6-oxo-5,6-dihydro-4Himidazo[1,5-a][1,4]benzodiazepine-3-carboxylate
Reference solution (b). Dissolve 2.5 mg of
(demethylflumazenil) with iodo[11C]methane or [11C]methyl demethylflumazenil R in 50 ml of methanol R.
trifluoromethanesulphonate.
Reference solution (c). To 0.1 ml of reference solution (a)
add 0.1 ml of reference solution (b) and dilute to V with a
Synthesis of iodo[11C]methane
Iodo[11C]methane may be produced from [11C]carbon dioxide 0.9 g/l solution of sodium chloride R, V being the maximum
recommended dose in millilitres.
or from [11C]methane. The most frequently used method is
Reference solution (d). Dilute 0.1 ml of reference solution (a)
reduction of [11C]carbon dioxide with lithium aluminium
to 50 ml with methanol R. Dilute 1.0 ml of this solution to V
hydride. The [11C]methanolate formed is reacted with
with a 0.9 g/l solution of sodium chloride R, V being the
hydriodic acid. Alternatively [11C]methane, either obtained
directly in the target or by on-line processes from [11C]carbon maximum recommended dose in millilitres.
dioxide, is reacted with iodine.
Column :
— size : l = 0.15 m, Ø = 3.9 mm,
Synthesis of [11C]methyl trifluoromethanesulphonate
11
[ C]methyl trifluoromethanesulphonate may be
— stationary phase : spherical octadecylsilyl silica gel for
prepared from iodo[11C]methane using a solid support
chromatography R (5 µm) with a specific surface area of
such as graphitised carbon, impregnated with silver
440 m2/g, a pore size of 100 nm and a carbon loading of
trifluoromethanesulphonate.
19 per cent,
General Notices (1) apply to all monographs and other texts
825