Journal of Radioanalytical and Nuclear Chemistry, Articles, Vol. 152, No.

1 (1991) 81-86

RAPID AND SENSITIVE DETERMINATION

OF PERTECHNETATE IN 9 9 Mo/9 9 m Tc GENERATOR ELUATES
BY REVERSED-PHASE HIGH-PERFORMANCE
LIQUID CHROMATOGRAPHY

R. A M A N O

School of Allied Medical Professions, Kanazawa University, Kodatsuno, Kanazawa 920 (JapanJ

(Received August 2, 1990)

Reversed-phase high-performance liquid chromatography with u.v. detection was applied

for rapid and sensitive determination of pertechnetate in 99 M0199mTc generator eluates,
using a mixture solvent of acetonitrile and 0.04M aqueous acetate buffer (1 l1 ) containing a
few volume percentage of 0.5M tetra-n-butylammonium hydroxide as the mobile phase.
Employing a /s-bondapak Cl8 column, the TcO~ species was separated, monitored with
absorbance at 254 nm, and observed at the retention time of 3.5 min. The detection limit
was found to be 5.2 9 10 -~ o g of Te for each injection. Total Tc contents in the 99mTc
eluates from clinically-used 99 MO]99mTc generator were analyzed by this technique. The
99 mTc (99 Tc) species was separated from the contaminant 99 Mo. This method was found
to be useful for the purification of 99 mTc (99 TC) as well as the determination of total Tc
content.

Introduction

Knowledge of the total concentration of pertechnetate (99 m Tc04 and 99 T c 0 4 )

in 99Mo/99mTc generator eluates is important in the quality control of 99mTc
radiopharmaceuticals) -4 High concentrations of technetium in the eluates can have
deleterious effects on the labeling efficiency and the imaging quality of certain
99 m Tc radiopharmaceuticals. The amount of 99 Tc has been experimentally estimated
by the measurement of 99 Tc radioactivity. Several calculation methods have been
published to evaluate the amount of 99 Tc in the 99 m Tc eluate. The calculated
values are not in agreement with the experimental values because of the difficulty
in assuming some important parameters (history of the 99M0 used for generator
preparation, elution efficiency etc.) in the calculation. HOLLAND et al. s,6 have
comprehensively investigated on eight commercially available generators by the use
of five different analytical procedure, and described that the observed Tc/99mTc
values were higher than the expected values. Therefore, it is necessary to determine
rapidly and directly the total pertechnetate contents in the eluates. ZODDA et al. 7

Elsevier Sequoia S. A., Lausanne

Akadgrniai Kiad6, Budapest
 R. AMANO: RAPID AND SENSITIVE DETERMINATIONOF PERTECHNETATE

had examined quantitative determination of pertechnetate by ion exchange high-

performance liquid chromatography and described that the detection limit of
K99TcO4 was 1.30 9 10-7M.
In this paper, we developed reversed-phase high-performance liquid chromatography
(reversed-phase HPLC) for the rapid and more sensitive determination of total per-
technetate in clinically used 99 m TcO4 eluate.

Experimental
Radioactive sources and chemicals

99Tc was purchased from the Amersham International Company as an aqueous

solution of ammonium pertechnetate. The 99 TcO4 standard solution was prepared
from this solution and the 99 Tc content was determined by liquid scintillation
counter which was calibrated using a weighable Rb compound. The 99 Tc concentra-
tion in the standard solution varied in the range between Bq/ml and MBcl/ml. 99mTc
generators, which were purchased from Daiichi Radioisotope Institute for clinical
purpose, were used within the available period. Eluting procedure was run according
to the recommended specification.
The MoO~- and ReO~ standard solutions were prepared from ammonium molybdate
tetrahydrate and ammonium perrhenate, respectively, which were of an extremely
puregrade. 0.5M tetra-n-butylammonium hydroxide (hereafter abbreviated as
TBA§ -) solution was purchased and used a s a HPLC solvent by diluting to other
solvents. All other chemicals used were of an extremely pure grade.

HPLC m e t h o d

HPLC analyses were performed on a reversed phase/~-bondapak C~s column

(Waters Co.) using as mobile phase a mixture solvent of acetonitrile and 0.04M
aqueous acetate buffer (pH 4.4) (1/1) containing a few volume percent of 0.5M
TBA*OH- solution. Injected volumes were 10-100 #1 for the standard solutions and
100-300/al for generator eluate. Flow rate was constant, 1 ml/min, for all experi-
ments. Retention time could be determined from the peak of u.v. absorption and/or
radioactivity. Absorbance was monitored at 254 nm by u.v. detector. Absorption
peak area was calculated by Chromatopac C - R 3 A (Shimadzu Co.). The 99 m Tc
radioactivities for the eluate were also monitored successively by ionization
chamber. Several 0.5 ml portions of eluate were also collected and analyzed for
their radioactive impurities by 3,-ray spectrometry using pure Ge semiconductor
detector.

82
 R. AMANO:RAPID AND SENSITIVE DETERMINATION OF PERTECHNETATE

Results and discussion

We initially examined the ion pair effect of TBA§ - on the retention times of
99TCO4, ReO; and MoO~-. Figure 1 shows the relationship obtained between the
retention times and the volume percentage of 0.5M TBA§ - solution added into a
mixture solvent of acetonitrile and 0.04M aqueous acetate buffer (pH 4.4) (1/1).
The solvent containing 2 - 3 vol.% of 0.5M TBA*OH- solution was found to give the

E
E
r
.o

cr

2i

L I I -D.
0 1 2 3
Vol. % of 0.5M
Fig. 1. Relationships between the volume percentage of 0.5M tetra-n-butylammortium hydroxide
and the retention times of 99TcO~ (o), ReO~ (~x)and MoO~"(t~)

appropriate retention times to separate each species and to avoid impurities. In fact,
the mixture of 99TCO4, ReO~ and MOO]- could be separated from each other, as
is shown in Fig. 2, using a HPLC solvent of acetonitrile and 0.04M aqueous acetate
buffer (pH 4.4) (1/1) containing 2 vol.% of 0.5M TBA§ - solution. The peaki of
MoO~-, ReO~ and TcO4 are given at the retention time of 1.9, 3.1 and 3.5 minutes,
respectively. No significant change of these retention times was observed in less than
100 gl injection volume.
The sensitivity of this HPLC system was examined for 99TcO4 ion. Figure 3
shows that the calibration curve is linear in the range of 5.2 9 10-4--1 9 10 -2 #g of
99Tc. The detection limit of 99Tc concentration was found to l~e 5.2 9 10 -9 g/ml,
which corresponds to 3.27 Bq/ml, for 100 gl injection volume, and 1.73 9 10 -9 g/ml,
which correspond to 1.09 Bq/ml, for 300 gl injection volume. Our detection limit
was much smaller than the published limit. I

83
R. AMANO: RAPID AND SENSITIVE DETERMINATION OF PERTECHNETATE

b c

I j J ~
6 8
Time, rain

Fig. 2. Elufion profile for the mixture of 99 TcO~, (c), ReO~ (b) and MOO2,- (a) using a solvent
of acetonitrile and 0.04M aqueous acetate buffer (pH 4.4) containing 2% volume of 0.5M
tetra-n-butylammonium hydroxide solution. The concentrations of Tc, Re and Mo were
0.15, 0.4 and 0.2 #g/ml, respectively

~5
•

2 4 6 8 10
Tc content, xlO"9 g

Fig. 3. Calibration curve for TcO~ ion analysis between the Tc contents and peak area

By the above experiments, it was demonstrated that the C18 column HPLC
chromatogram of a small amount of 99TCO4 with a mixture solvent of acetonitrile
and aqueous acetate buffer solution containing TBA*OH - provides a direct and rapid
method of determining the total concentration of pertechnetate (99 m TcO4 and
99TcO~) in the 99Mo/99mTc generator eluates.

84
 R. AMANO: RAPID AND SENSITIVE DETERMINATION OF PERTECHNETATE

99M~ t ~>>"

T~
4 u
\/-
0
99mTc
13

I
I
u

t)
t-
t~
i Too0
J
_ A U S

0 6 8
rime, rain
Fig. 4. Typical HPI..C profile of clinically-used generator eluate. Absorbance of ToO; and radio-
activity of 99 mTc were monitored by u.v. 254 nm and ionization chamber, respectively,
and radioactive impurity of 99 Mo was also determined as shown in the upper part

An aliquot ( 1 0 0 - 3 0 0 pl) of clinically used 99mTcO4 eluate was analyzed under

the above conditions. Typical chromatogram is shown in Fig. 4. The vibration at
the fast retention time of 1 . 3 - 1 . 8 minutes may be due to the fairly large amount o f
chloride ion, because clinically used 99mTcO4 is eluted in a physiological salt solu-
tion. A sharp peak was observed at a retention time of 3.5 minutes. Radioactivity
measurement by ionization chamber also showed a single major component with
retention time of 3.5 minutes, confirming that the component was indeed pertechnetate
ion. Several collected 0.5 ml portions were examined for the remaining radioactivity
after full decay of 99mTc. By Ge 7-ray spectrometry, 99Mo radioactive impurities
were observed in some portions as shown by a histogram in the upper part o f Fig. 4.
The absorbance peak at a retention time o f 2.0 minutes may be due to MoO~- ion.
The concentration of Tc( 99 Tc + 99 m Tc ) in the clinically used eluate was calculated
to be 0.57/ag/ml, which was a hundred times greater than the detection limit. In this

85
R. AMANO: RAPID AND SENSITIVE DETERMINATION OF PERTECHNETATE

experiment, more than 99% of initially injected radioactivity was recovered from
the HPLC column within the peak at retention time of 3.5 minutes. The radioactivity
concentration was also measured to be 1.68 GBq of 99mTc/ml of eluate. The
specific radioactivity was calculated to be 2.95 GBq of 99mTc//.tg of Tc. The total
impurity radioactivity of 99Mo was estimated to be 35 Bq, which corresponded to
7 kBq/20 ml of eluate. The impurities 99Mo are due to breakthrough from the
alumina generator column. The breakthrough percentage was calculated as 2 9 10-s%.
In general, the concentration of perteclmetate eluted from recent clinically used
generators was estimated to be in the range of 10 .9 g / m l - 1 0 -7 g/ml through their
useful clinical life. Therefore, it is evident that this technique has the rapidity,
sensitivity and seleotivity necessary to monitor total chemical pertechnetate in
actual 99Mo/99mTc generator eluates. Moreover this method was found to be useful
for the purification of 99mTc (99Tc) as well as the determination of total Tc
content.

This work was supported in paxt by Grants-in-Aid for Scientific Research from the Ministry
of Education, Science and Culture, Japan.

References

1. v. HUSAK, J. VLCEK, Int. J. Appl. Radiation Isotopes, 30 (1979) 165.

2. R. E. BOYD, Radiochim. Acta, 30 (1982) 123.
3. R. BAUER, H. PABST, Eur. J. Nucl. Med., 7 (1982) 35.
4. K. THIEME, Int. J. Appt. Radiation Isotopes, 39 (1988) 267.
5. M. E. HOLLAND, E. DEUTSCH, W. R. HEINEMAN, E. M. HOLLAND, Int. J. Appl. Radiation
Isotopes, 37 (1986)165.
6. M. E. HOLLAND, E. DEUTSCH, W. R. HEINEMAN, Int. J. Appl. Radiation Isotopes, 37
(1986) 173.
7. J. P. ZODDA, W. R. HEINEMANN, T. W. GILBERT, E. DEUTSCH, J. Chromatogr. 227 (1982)
249.

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