Appl. Rndiat. ht. Vol. 31, No. 7, pp. 593-598, 1986 0883-2889/86 $3.00 + 0.

00
ht. J. Radiat. Appl. Iawum. Part A Pergamon Journals Ltd
Printed in Great Britain

Reversed-Phase HPLC of 99mTc Methylene

Diphosphonate Bone Imaging Kits
with Quantification of Pertechnetate

DANIEL J. HOCH and THOMAS C. PINKERTON

Department of Chemistry. Purdue University. West Lafayette, IN 47907, U.S.A

Ion-pairing reversed-phase HPLC is used to separate a mixture of yrmTc methylene diphosphonate (MDP)
complexes prepared by tin reduction of pertechnetate in the presence of hgand. Chromatographic
conditions allow for the quantification of total pertechnetate concentration, as well as the determination
of chemical purity of generator eluents from which y9mTc is derived. Both determinations can be made
prior to actual labeling. Commercially available MDP kits from three manufacturers are evaluated. All
three MDP kits, when labeled with ‘9mT~, produce multiple, chromatographically separable components.
The formation of these TcMDP complexes is time dependent. The labeling procedure produces several
products within the first 20 min, with up to fifteen complexes observed after 4 h. The results of this work
demonstrate the presence of substantial chemical contamination in generator eluents.

Introduction different 99mTc-MDP complexes; however, the tech-

nique does not facilitate the isolation of separated
Commercial kits utilizing 99mT~ labeled diphosphon-
components for further investigation.“)
ates as bone imaging agents are widely used in the
High resolution separations of labeled 99mTc
field of diagnostic nuclear medicine.“,‘) These rddio-
diphosphonate mixtures have been achieved with
pharmaceutical kits contain a lyophilized mixture of
anion-exchange HPLC, utilizing polystyrene divinyl-
reductant and ligand. Commercial use of tin as the
benzene strong ion-exchange resins.“&‘4) In contrast
reductantJ3) and methylene diphosphonate (MDP)
to Tc-HEDP preparations, the Tc-MDP complexes
as the bone seeking ligand, is widely accepted.‘4) The
yield low recoveries when chromatographed on
labeling procedure is carried out by the addition of
strong anion-exchangers, thus contributing to non-
the eluent from a %Mo/~~“Tc generator to the kit.
reproducibility and decreased column life.‘6,“.‘“) In
The generator eluent contains [99mTc]pertechnetate in
addition, strong anion-exchange resins are not suited
normal saline. The addition of 99mTc0; to commer-
for the determination of total technetium concen-
,:ial diphosphonate kits is shown to yield a mixture of
tration because high affinity prevents the elution of
:nroducts at both the “carrier added” level as well as
TcO;. In view of this, it is necessary to use guard
!he “no carrier added” level.(s ‘) The general labeling
columns and back-flushing techniques to remove un-
,.eaction can be written as follows.
reacted pertechnetate from anion-exchange columns
in order to prevent on-column reactions when an
99mTc0; + Sn + MDP
active reductant, such as Sn2+, remains in the injected
-+99mTc(Sn)MDP (mixture of complexes) sample.(‘3’
The total technetium concentration in a generator
Previously, separation of product mixtures had eluent has been shown to vary with the age and
Teen obtained with paper or thin layer chromatog- history of a generator, as well as the time since
.aphy. (8,9)These low performance methods character- previous elution. (“.w Since pertechnetate cannot be
.stically distinguish only three components; y9mTc eluted, strong anion-exchange HPLC cannot be used
labeled product, unreduced pertechnetate, and to quantify the total amount of technetium (i.e. “Tc
hydrolyzed reduced pertechnetate. These quality and ““Tc) in the generator eluent prior to labeling.
control procedures lack the resolution necessary to Ideally. the chromatographic method should be suit-
evaluate the chemical nature of either the y9mTc able for both (i) the determination of total technetium
labeled compounds or the hydrolyzed reduced tech- concentration in a generator eluent and (ii) the
netium. An alternative separation method is electro- separation of labeled 99mTc-MDP complexes in a
phoresis. This has been shown to yield up to four resultant reaction mixture.

593
594 DANIEL J. HOCH and THOMAS C. PINKERTON

This work presents an ion-pairing reversed-phase radiochemical purity, Oak Ridge Laboratories, Oak
high performances liquid chromatographic separ- Ridge, Tenn.). The resulting potassium pertechnetate
ation of tin reduced 99mTc-MDP complexes on a was recrystallized twice to insure purity.
macroporous silica bonded reversed-phase column. Samples of 99mTc present in generator eluents were
The radiopharmaceutical kits are prepared at “no provided by a regional distributor of radiopharma-
carrier added” levels by manufacturer recommended ceutical products. The Tc-MDP bone imaging kits
procedures. The improved separation of the Tc(Sn) were obtained from several manufacturers.
MDP reaction, as well as the increased recovery
afforded by the use of ion-pairing reversed-phase Procedures
HPLC, enables the profiling of 99mTc-MDP prepara- The bone imaging kits were reconstituted accord-
tions and isolation of individual Tc-MDP complexes ing to manufacturers instructions. In each case
for subsequent characterization. Is also allows for the 20 mCi of 99mTc, in I mL of normal saline generator
quantification of pertechnetate and the determination eluent, was added to the commercial kit. The sample
of chemical purity in generator eluents prior to was allowed to sit for 20min before an aliquot was
labeling. removed for injection onto the chromatographic
column.
Experimental
Apparatus
Results and Discussion
The chromatographic equipment included a
ConstaMetric Model III (Laboratory Data Control) Quantljication of pertechnetate in generator eluents
dual piston pump, a Rheodyne Model 7125 injection Solutions of pure, recrystallized pertechnetate
valve with a 100 PL loop. The analytical column (99Tc0; ) were subjected to ion-pairing reversed-
consisted of a 250 mm L x 4 mm i.d. stainless steel phase HPLC to determine retention time and percent
column containing a macroporous C-18 reversed- recovery. Detection was accomplished by the use of
phase packing (Vydac), with particle diameter of a fixed wavelength U.V. detector at 254 nm. Samples
IO pm and 3OOA pore size. prepared in water yielded a single peak with a
Detection methods included the use of an retention time of 10.5 min and a recovery of > 98%.
aluminum housed NaI(TI) well scintillation crystal Since the 99mTc obtained from the technetium
(Harshaw, type 75F8) to detect radiation from the generator is supplied in normal saline (0.9% NaCI),
99mTc (140 KeV yray tllz = 6.0 h) upon elution from standards were also prepared in saline. Injection of
the HPLC column. An Ortec model 420A timing these samples yielded a split peak for pertechnetate
single channel analyzer, Harshaw NA-23 amplifier, (Fig. I). Through the use of a photodiode array
and a Harshaw NR linear ratemeter comprised the y u.v.-visible spectrophotometric detector, full wave-
detection electronics. A fixed wavelength Altex model length (20~-8OOnm) spectra were obtained during
153 u.v.-visible liquid chromatography detector was chromatographic elution. Both peaks exhibited
utilized in-line after the scintillation detector. The spectra identical to that of pertechnetate. The peak
fixed wavelength detector was operated at 254nm. splitting observed appeared to be a chromatographic
artifact caused by the large concentration of sodium
Materials chloride present in the sample, which tie up available
The mobile phase consisted of HPLC grade sodium ion-pairing reagent. This overloading of the ion-
acetate (Fisher Scientific), anhydrous reagent grade pairing reagent causes a portion of the pertechnetate
ethyl acetate (MCB Reagents) and tetrabutyl- band to initially move ahead on the column. This
ammonium hydroxide (Aldrich). All solutions were phenomenon has not been reported by previous
prepared with distilled, deionized water which had investigators using ion-pairing reversed-phase for the
been purified by a mixed bed ion-exchanger and separation of MDP kits. (6) Elimination of the peak
0.2pm filter with circulation through an activated splitting artifact was accomplished by an appropriate
carbon bed in a Water-I unit (Gelman Sciences). dilution of the pertechnetate sample in the mobile
Final concentrations of the mobile phase constituents phase. The minimum dilution required was deter-
were 1 x lO_lM sodium acetate, 2 x IO-‘M tetra- mined empirically to be a 2: 3 mixture of mobile phase
butylammonium hydroxide, and 3% (v/v) ethyl to sample (Fig. I). All subsequent samples which
acetate. The pH was adjusted to 6.0 with acetic acid. contained saline were diluted in this manner.
In house normal saline samples were prepared with Utilizing the 254 nm fixed wavelength detector,
certified A.C.S. sodium chloride (Fisher Scientific) calibration curves were constructed by measuring
diluted in distilled, deionized water further purified chromatographic peak heights as a function of total
by the Water-I. Samples of normal saline commonly pertechnetate (i.e. 99T~04 and 99”T~04) concen-
employed to elute technetium generators were tration. Over the “no carrier added” concentration
obtained from several manufacturers. range of I x IO-’ M to 5 x 10m6M a linear response
Potassium pertechnetate was obtained by the was observed with a correlation coefficient of
metatheses of ammonium pertechnetate (99% 0.999. At higher “carrier added” pertechnetate
 Reversed-phase HPLC of WmT~-MDP 595

A
_1:
(a) ( b)

L
I I I I I I
0 6 16 0 a 16 0 6 16

min

Fig. 1, Ion-pairing reversed-phase HPLC of 1O-6 M TcO; standards prepared in normal saline. Diluted
by various degrees in mobile phase, (a) no dilution, (b) 4: 1 sample to mobile phase, (c) 3:2 sample to
mobile phase. Mobile phase 3% ethyl acetate, IO-) M sodium acetate, 10m3M TBAOH, pH 6;
column = C-18 macroporous; U.V. detection at 254 nm; flow rate = 0.35 mL/min; sample size 100 PL; (0)
denotes detection artifacts of sample solvent front.

concentrations, a linear response was observed up to cial normal saline used to elute the generator. The
I x 10m4M. number and relative magnitude of the unknown
A concurrent method to quantify total pertechne- components are similar to that of the generator
tate concentration is liquid scintillation counting.“‘,‘*) eluent. Figure 5 shows a chromatographic separation
This requires at least a 60 h delay (10 half-lives) to of normal saline prepared with certified A.C.S. so-
(ensure complete decay of 99mTc to 99Tc. The HPLC dium chloride in distilled/deionized/carbon filtered
:method described herein utilizes optical detection to water. The first two peaks are solvent front artifacts.
[quantify the total pertechnetate and therefore can be No responses were detected beyond 10min.
Iused immediately on samples containing 99mTc. The The samples of [99mTc]pertechnetate generator elu-
,:oncentration of pertechnetate in generator eluent ent were studied over the clinically useful lifetime of
samples, was determined by both procedures. Results two separate generators from the same manufacturer.
If the measured concentrations from both methods Both generators were eluted with the same saline and
.tre compared graphically in Fig. 2. A linear corre- both indicated the presence of U.V. absorbing im-
ation coefficient of 0.973 was obtained between the
~nethods.

Chemical impurities 3
Upon HPLC of [99mTc]pertechnetate generator
ramples, one major peak was observed in the y trace
i
while a large number of contaminates were observed
I ./
with U.V. detection (Fig. 3). The retention times of the 2- . l

1 peak, as well as a corresponding U.V. peak, were ^r

4
identical to that of pertechnetate. The remaining U.V.
absorbing components were attributed to chemical I:
a
i npurities in the generator eluent. Deutsch et a/.,(‘61 I
t - ./
I-ave reported the presence of a large number of U.V.
absorbing impurities present in generator eluents. / .
l’hese impurities were presumed to be leached from
t,le plastic lining of the generator column. In this
s’ udy the source of the impurities was found to be in
1 /-
I I I
the normal saline used to elute the generator and not 1 2 3
the plastic column lining. This is in agreement with LSC (FM)
the findings of Boyd,“‘) who also reported the pres-
Fig. 2. Correlation of total pertechnetate concentration as
ence of U.V. absorbing impurities in normal saline. determined by both HPLC and liquid scintillation counting.
Figure 4 illustrates a chromatograph of the commer- Slope = 1.01; correlation coefficient = 0.973.
596 DANIEL J. How and THOMAS C. PINKERTON

purities
Fig. 3. Ion-pairing

which increased
1
-----
0
?I! i

reversed-phase
detection, __

with
10

generator
I

1,
1’
/; k---___

20

HPLC of generator

age. Figure
Time

6
30

Imln)

eluent. Conditions
uv. detection); (*) pertechnetate,
40

same as Fig.
(a) artifacts.
50

1: (-----) y-ray

reported.““,2’) This is believed to be an infrequent

illustrates the chromatographic elution of [““‘Tc]per- phenomenon and we have no evidence to suggest
technetate generator samples on days 1, 3, and 6. The such occurred with the limited number of generators
starred peak indicates pertechnetate. studied.
The U.V. impurities may affect ““‘Tc labeling.
Evidence for this is seen in Fig. 7 which shows both
the U.V. and y chromatographs for a ““‘Tc labeled Separation of Tc-MDP mixtures
commercial MDP kit. The starred peaks in the trace, The MDP radiopharmaceutical kits provided by
which contain y9mT~, have retention times corre- three manufacturers were all prepared identically and
sponding to impurity peaks observed in the U.V. aliquots were taken at timed intervals for chro-
chromatogram of the generator eluent. This infers the matographic evaluation. All three manufacturers’
99mTc labeling of impurities. products resulted in mixtures of chromatographically
The presence of radiochemical impurities (e.g. separable 99mT~-MDP complexes, as illustrated in Fig.
“‘Cs, ‘“‘Ru, etc.) in the generator eluent has been 8. Previous findings by Srivastava,‘@ demonstrated up
to five Tc(Sn)MDP components by reversed-phase
ion-pairing chromatography. A greater number of
.

0002au . .
i

0
l d!.l:: I
10

Fig. 4. Ion-pairing
I
20
Time

reversed-phase
( mm)
I
30

HPLC of commercial
40
I I
50
_1
-0
- I

Fig. 5. Ion-pairing
,
10
_J_

I
20
min

reversed-phase
-._l

30
I

HPLC of normal saline

I
40

normal saline used to elute generator: conditions same as prepared with certified A.C.S. sodium chloride in purified
Fig. I: U.V. detection at 254 nm: (e) artifacts. water; u.v. detection at 254 nm; (0) artifacts.
 Reversed-phase HPLC of 9YmTc-MDP 597

99mTc-MDP complexes can be observed in Fig, 8.

This is attributed to the macroporous reversed-phase
support used in this study. The larger pore diameter
(300 A) enables a greater partitioning phase
accessibility for suspected oligomeric 9’mTc-MDP
complexes.
At extended reaction times more y9mTc-MDP com-
ponents are produced, Fig. 9. It is believed that these
complexes are polymeric species which form only
after a suitable time period. Wilson and Pinkerton’m
have demonstrated the existence of technetium
diphosphonate polynuclear complexes with analog
Tc(NaBH,)HEDP preparations by determining the
charge and size of 12 Tc-HEDP complexes by anion-
exchange HPLC. The charges ranged from - 1.5 to
-8.0 with partial molar volumes of 510-1600
mL/mol. (I’) Mixed metal technetium diphosphonate
complexes containing tin have also been suggested by
Van den Brand.@?’

Conclusion
The MDP labeling kits, provided by three manu-
facturers, all yielded multiple chromatographically
separable Y9mTc-MDP complexes. Ion-pairing
reversed-phase chromatography was demonstrated as
an excellent means of separating these mixtures. The
HPLC procedure is suitable for the quantification of
total technetium concentration and the detection of
chemical impurities in generator eluents. The source
of U.V. absorbing impurities in generator eluents was
found to be in the normal saline used to elute a
generator.
, uu , I I I I
0 10 20 30 40 so

min

Fig. 6. Ion-pairing reversed-phase HPLC of generator Acknowledgements-This work was supported in part by
eluent; conditions same as Fig. 1: (*) pertechnetate; Public Health Service Grant RNM ROl-CA40110-1 award
(0) artifacts; samples of generator eluent eluted on days; by the National Cancer Institute, Department of Health
(a) = 1, (b) = 3, and (c) = 6; U.V. detection at 254 nm. and Human Services.

I
U I I 1 I

0 10 20 30 40 50

Fig. 7. Ion-pairing reversed-phase HPLC of Tc(Sn)MDP preparation; conditions same as Fig. 1: (*)
denotes impurities in saline which have been labeled with 99mTc; (-- u.v. detection, y-ray detection).
598 DANIEL J. HOCH and THOMASC. PINKERTON

(a) (b)
I

0 4 a !O 12 0 4 610 0 a 16 24

mln

Fig. 8. Ion-pairing reversed-phase HPLC of commercial Tc(Sn)MDP preparations, manufactures a, b, and

c; all samples are 20 min post formulation; conditions same as Fig. 1, except flow rate in a and b is
0.6 mL/min; y-ray detection.

1 I I I I
0 8 16 24 30

J I I I I
0 8 16 24 30

Fig. 9. Ion-pairing reversed-phase HPLC of commercial Tc(Sn)MDP at (a) = 20 min, (b) = 3 h, and
(c) = 4 h post formulation; conditions same as Fig. I: y-ray detection.

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