Radiochemical purity control of radiopharmaceutical
As with other drugs, there are certain claims posed also on radiopharmaceuticals. Quality
requirements on drugs valid in EU are liste in European Pharmacopoeia.
Radiochemical purity is in Ph.Eur. defined as the ratio, expressed as a percentage, of the
activity of the radionuclide concerned which is present in the radiopharmaceutical preparation
in stated chemical form, to the total radioactivity of that radionuclide present in the
radiopharmaceutical preparation. The relevant radiochemical impurities are listed with their
limits in the individual monographs. The requirement of the radiochemical purity must be
fulfilled throughout the period of validity.
Radiochemical impurities may origine from:
- radionuclide production
- subsequent chemical procedures
- incomplete preparative separation
- chemical changes during storage
Determination of radiochemical purity requires separation of the different chemical
substances containing raionuclide and estimating the percentage of radioactivity associated
with the declared chemical substance. In principle, any method of analytical separation may
be used. For example, the monographs for radiopharmaceutical products may include paper
chromatography, thin-layer chromatography, electrophoresis, size-exclusion chromatography,
gaz chromatography and liquid chromatography.
In a hospital environment thin-layer and paper chromatography are mostly used. In paper
and thin-layer chromatography, a volume equal to that described in the monograph is
deposited on the starting-line. After development the support is dried and the positions of the
radioactive areas are detected by autoradiography or by measurement of the radioactivity over
the length of the chromatogram, using suitable collimated counters or by cutting strips and
counting each portion. The positions of the spots or areas permit chemical identification by
comparison with the solutions of the same chemical substance (no-radioactive) using
a suitable detection method.
Procedure: Apply defined volume (according to the monographs, most usually 3-5 μl) of the
radiopharmaceutical preparation on the strip of chromatographic paper or the plate (usual
parameters 2,5 x 15 cm for ascending or 2,5 x 25 cm for descending chromatography) with
the aid of syringe or micropipette. In case of exceeding activity of the radiopharmaceutical
preparation, the is a way of dilution described in monograph. Let the solution dry, the put it in
to the chromatographic tank. Develop immediately over a path (usually of 10 cm to 20 cm)
filled with eluent solution. Let the plate dry. Place dried chromatogram into the
chromatographic ruler between two folia strips which prevent contamination in the way that
the start is on 11 cm of the chromatographic ruler. Insert the ruler into the lead shield of the
detector. The gap of the detector is directly above the start of the chromatogram (Rf = 0).
Width of the detector gap esure that only the narrow segment of the chromatogram is
measured at once. Move the ruler inside the detector and measure the distribution of the
activity along the chromatogram. Proceed by 1 cm and measure the counts along all
chromatogram. Impulses generated by the detector whose frequency is dependent on the
activity are registered by spectrometric device. Positions of spots and their Rf values are
determined by comparison with the chromatograms of standars containig radionuclide or by
the comparison with the loaction of the spots of non-radioactive substances (developped in the
same conditions as the radiopharmaceutical preparation).
Example of chromatogram evaluation:
Rf
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
counts (imp./60 s)
380
126
22
16
13
15
17
18
46
138
382
1236
2836
792
206
173
89
73
48
17
16
Paper chromatography:
eluent
methanol:water (80:20)
99m
Na TcO4 Rf = 0,6
99m
TcO2
Rf = 0,0
Radiochemical purity control of Sodium pertechnetate (99mTc)
injection (non-fission) by paper chromatography
Radiochemical purity control of Sodium pertechnetate (99mTc)
injection (fission) by paper chromatography
Task 1
Radiochemical purity control (Ph. Eur):
descending paper chromatography
eluent: water:methanol (20:80)
Na99mTcO4
99m
TcO2
Rf = 0,6
Rf = 0,0 (reduced hydrolysed forms of technetium)
Procedure: Apply 5 µl (1 drop) of the preparation with aid of a syringe on the
chromatographic paper Whatman 3. After drying develop instantly for 2 h using a mixture of
20 volumes of water and 80 volumes of methanol along the path of 10 cm and more. Allow
the paper to dry. Determine the distribution of radioctivity using a suitable detector. Measure
by 0,5 cm 3 times. Calculate the average of the counts. Draw a graph of dependency between
Rf and counts. Identify radiochemical impurities and the main product.
%peak A = 100 x
sum of counts measured for the peak A
sum of counts for all chromatogram
%peak B = 100 x
sum of counts measured for the peak B
sum of counts for all chromatogram
Not less than 95 % of the total radioactivity is in the spot corresponding to per technentate
ion, which has an Rf value of about 0,6.
Task 2
Rf
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
0,45
99m
TcO4-
kit (sodium
acetate)
kit (2butanone)
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
Radiochemical purity control of Sodium iodohippurate (131I)
injection by thin-layer chromatography
Not less than 96 % of the iodine-131 is in the form of sodium 2-iodohippurate.
Radiochemical purity control (Ph. Eur):
thin-layer chromatography using silica gel GF254 as the coating substance
eluent: mixture of water (1 volume) : glacial acetic acid (4 volumes) : butanol (20 volumes) :
toluene (80 volumes)
iodine ion
Rf = 0,0-0,1
2-iodohippuric acid Rf = 0,5-0,6
2-iodobenzoic acid Rf = 0,9-1,0
Procedure:
Test solution: Dissolve 1 g of potassium iodide in 10 ml of water, add 1 volume of this
solution to 10 volumes of the injection to be examined and use within 10 min of mixing. If
necessary dilute with the reference solution (carrier) to give a radioactive concentration
sufficient for the detection method.
Reference solution (carrier): Dissolve 40 mg of 2-iodohippuric acid and 40 mg of 2iodobenzoic acid in 4 ml of 0,1 M sodium hydroxide, add 10 mg of potassium iodide and
dilute to 10 ml with water.
Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm (about 75
min) using a mixture of 1 volume of water, 4 volumes of glacial acetic acid, 20 volumes of
butanol and 80 volumes of toluene. Allow the plate to dry in air and examine in ultraviolet
light at 254 nm. The chromatogram obtained with the reference solution shows a spot
corresponding to 2-iodohippuric acid and nearer to the solvent front a spot corresponding to
2-iodobenzoic acid. Iodine ion remains near the starting line. Determine the distribution of
radioactivity using a suitable detector. In the chromatogram obtained with the test solution,
not less than 96 % of the total radioactivity is found in the spot corresponding to 2iodohippuric acid and not more than 2 % of the total radioactivity is found in either of the
spots corresponding to 2-iodobenzoic acid and to iodine ion.
Sample No.1
Rf
1.
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Sample No.2
counts (imp./60 s)
2.
3.
Rf
average
1.
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
counts (imp./60 s)
2.
3.
average