- Email: [email protected]
Reversed-phase HPLC of 99mTc methylene diphosphonate bone imaging kits with quantification of pertechnetate
Appl. Rndiat. ht. Vol. 31, No. 7, pp. 593-598, ht. J. Radiat. Appl. Iawum. Part A Printed in Great Britain
1986
0883-2889/86 $3.00 + 0.00 Pergamon Journals Ltd
Reversed-Phase HPLC of 99mTc Methylene Diphosphonate Bone Imaging Kits with Quantification of Pertechnetate DANIEL Department
J. HOCH
of Chemistry.
and
Purdue
THOMAS
University.
C. PINKERTON
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 kits utilizing 99mT~ labeled diphosphonates as bone imaging agents are widely used in the field of diagnostic nuclear medicine.“,‘) These rddiopharmaceutical kits contain a lyophilized mixture of reductant and ligand. Commercial use of tin as the reductantJ3) and methylene diphosphonate (MDP) as the bone seeking ligand, is widely accepted.‘4) The labeling procedure is carried out by the addition of the eluent from a %Mo/~~“Tc generator to the kit. The generator eluent contains [99mTc]pertechnetate in normal saline. The addition of 99mTc0; to commer,:ial diphosphonate kits is shown to yield a mixture of :nroducts at both the “carrier added” level as well as !he “no carrier added” level.(s ‘) The general labeling ,.eaction can be written as follows. Commercial
99mTc0; + Sn + MDP -+99mTc(Sn)MDP
(mixture
of complexes)
Previously, separation of product mixtures had Teen obtained with paper or thin layer chromatog.aphy. (8,9)These low performance methods character.stically distinguish only three components; y9mTc labeled product, unreduced pertechnetate, and hydrolyzed reduced pertechnetate. These quality control procedures lack the resolution necessary to evaluate the chemical nature of either the y9mTc labeled compounds or the hydrolyzed reduced technetium. An alternative separation method is electrophoresis. This has been shown to yield up to four 593
different 99mTc-MDP complexes; however, the technique does not facilitate the isolation of separated components for further investigation.“) High resolution separations of labeled 99mTc diphosphonate mixtures have been achieved with anion-exchange HPLC, utilizing polystyrene divinylbenzene strong ion-exchange resins.“&‘4) In contrast to Tc-HEDP preparations, the Tc-MDP complexes yield low recoveries when chromatographed on strong anion-exchangers, thus contributing to nonreproducibility and decreased column life.‘6,“.‘“) In addition, strong anion-exchange resins are not suited for the determination of total technetium concentration because high affinity prevents the elution of TcO;. In view of this, it is necessary to use guard columns and back-flushing techniques to remove unreacted pertechnetate from anion-exchange columns in order to prevent on-column reactions when an active reductant, such as Sn2+, remains in the injected sample.(‘3’ The total technetium concentration in a generator eluent has been shown to vary with the age and history of a generator, as well as the time since previous elution. (“.w Since pertechnetate cannot be eluted, strong anion-exchange HPLC cannot be used to quantify the total amount of technetium (i.e. “Tc and ““Tc) in the generator eluent prior to labeling. Ideally. the chromatographic method should be suitable for both (i) the determination of total technetium concentration in a generator eluent and (ii) the separation of labeled 99mTc-MDP complexes in a resultant reaction mixture.
DANIEL J. HOCH and THOMAS
594
This work presents an ion-pairing reversed-phase high performances liquid chromatographic separation of tin reduced 99mTc-MDP complexes on a macroporous silica bonded reversed-phase column. The radiopharmaceutical kits are prepared at “no carrier added” levels by manufacturer recommended procedures. The improved separation of the Tc(Sn) MDP reaction, as well as the increased recovery afforded by the use of ion-pairing reversed-phase HPLC, enables the profiling of 99mTc-MDP preparations and isolation of individual Tc-MDP complexes for subsequent characterization. Is also allows for the quantification of pertechnetate and the determination of chemical purity in generator eluents prior to labeling.
C.
PINKERTON
radiochemical purity, Oak Ridge Laboratories, Oak Ridge, Tenn.). The resulting potassium pertechnetate was recrystallized twice to insure purity. Samples of 99mTc present in generator eluents were provided by a regional distributor of radiopharmaceutical products. The Tc-MDP bone imaging kits were obtained from several manufacturers. Procedures The bone imaging kits were reconstituted according to manufacturers instructions. In each case 20 mCi of 99mTc, in I mL of normal saline generator eluent, was added to the commercial kit. The sample was allowed to sit for 20min before an aliquot was removed for injection onto the chromatographic column.
Experimental Apparatus The chromatographic equipment included a ConstaMetric Model III (Laboratory Data Control) dual piston pump, a Rheodyne Model 7125 injection valve with a 100 PL loop. The analytical column consisted of a 250 mm L x 4 mm i.d. stainless steel column containing a macroporous C-18 reversedphase packing (Vydac), with particle diameter of IO pm and 3OOA pore size. Detection methods included the use of an aluminum housed NaI(TI) well scintillation crystal (Harshaw, type 75F8) to detect radiation from the 99mTc (140 KeV yray tllz = 6.0 h) upon elution from the HPLC column. An Ortec model 420A timing single channel analyzer, Harshaw NA-23 amplifier, and a Harshaw NR linear ratemeter comprised the y detection electronics. A fixed wavelength Altex model 153 u.v.-visible liquid chromatography detector was utilized in-line after the scintillation detector. The fixed wavelength detector was operated at 254nm. Materials The mobile phase consisted of HPLC grade sodium acetate (Fisher Scientific), anhydrous reagent grade ethyl acetate (MCB Reagents) and tetrabutylammonium hydroxide (Aldrich). All solutions were prepared with distilled, deionized water which had been purified by a mixed bed ion-exchanger and 0.2pm filter with circulation through an activated carbon bed in a Water-I unit (Gelman Sciences). Final concentrations of the mobile phase constituents were 1 x lO_lM sodium acetate, 2 x IO-‘M tetrabutylammonium hydroxide, and 3% (v/v) ethyl acetate. The pH was adjusted to 6.0 with acetic acid. In house normal saline samples were prepared with certified A.C.S. sodium chloride (Fisher Scientific) diluted in distilled, deionized water further purified by the Water-I. Samples of normal saline commonly employed to elute technetium generators were obtained from several manufacturers. Potassium pertechnetate was obtained by the metatheses of ammonium pertechnetate (99%
Results and Discussion Quantljication
of pertechnetate
in generator
eluents
Solutions of pure, recrystallized pertechnetate (99Tc0; ) were subjected to ion-pairing reversedphase HPLC to determine retention time and percent recovery. Detection was accomplished by the use of a fixed wavelength U.V. detector at 254 nm. Samples prepared in water yielded a single peak with a retention time of 10.5 min and a recovery of > 98%. Since the 99mTc obtained from the technetium generator is supplied in normal saline (0.9% NaCI), standards were also prepared in saline. Injection of these samples yielded a split peak for pertechnetate (Fig. I). Through the use of a photodiode array u.v.-visible spectrophotometric detector, full wavelength (20~-8OOnm) spectra were obtained during chromatographic elution. Both peaks exhibited spectra identical to that of pertechnetate. The peak splitting observed appeared to be a chromatographic artifact caused by the large concentration of sodium chloride present in the sample, which tie up available ion-pairing reagent. This overloading of the ionpairing reagent causes a portion of the pertechnetate band to initially move ahead on the column. This phenomenon has not been reported by previous investigators using ion-pairing reversed-phase for the of the peak separation of MDP kits. (6) Elimination splitting artifact was accomplished by an appropriate dilution of the pertechnetate sample in the mobile phase. The minimum dilution required was determined empirically to be a 2: 3 mixture of mobile phase to sample (Fig. I). All subsequent samples which contained saline were diluted in this manner. Utilizing the 254 nm fixed wavelength detector, calibration curves were constructed by measuring chromatographic peak heights as a function of total pertechnetate (i.e. 99T~04 and 99”T~04) concentration. Over the “no carrier added” concentration range of I x IO-’ M to 5 x 10m6M a linear response was observed with a correlation coefficient of 0.999. At higher “carrier added” pertechnetate
Reversed-phase HPLC of WmT~-MDP
595
_1: A ( b)
(a)
I
I
I
I
0
6
16
0
L
I
I
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 IO-) M sodium acetate, 10m3M TBAOH, pH 6; mobile phase. Mobile phase 3% ethyl acetate, 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 I x 10m4M. A concurrent method to quantify total pertechnetate concentration is liquid scintillation counting.“‘,‘*) This requires at least a 60 h delay (10 half-lives) to (ensure complete decay of 99mTc to 99Tc. The HPLC :method described herein utilizes optical detection to [quantify the total pertechnetate and therefore can be Iused immediately on samples containing 99mTc. The ,:oncentration of pertechnetate in generator eluent samples, was determined by both procedures. Results If the measured concentrations from both methods .tre compared graphically in Fig. 2. A linear correation coefficient of 0.973 was obtained between the ~nethods. Chemical impurities
cial normal saline used to elute the generator. The number and relative magnitude of the unknown components are similar to that of the generator eluent. Figure 5 shows a chromatographic separation of normal saline prepared with certified A.C.S. sodium chloride in distilled/deionized/carbon filtered water. The first two peaks are solvent front artifacts. No responses were detected beyond 10min. The samples of [99mTc]pertechnetate generator eluent were studied over the clinically useful lifetime of two separate generators from the same manufacturer. Both generators were eluted with the same saline and both indicated the presence of U.V. absorbing im-
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 ^r 1 peak, as well as a corresponding U.V. peak, were 4 identical to that of pertechnetate. The remaining U.V. I: absorbing components were attributed to chemical a I i npurities in the generator eluent. Deutsch et a/.,(‘61 ./ 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 I s’ udy the source of the impurities was found to be in I I 1 2 3 the normal saline used to elute the generator and not the plastic column lining. This is in agreement with LSC (FM) the findings of Boyd,“‘) who also reported the presFig. 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 commerSlope = 1.01; correlation coefficient = 0.973.
1 /-
and THOMAS C. PINKERTON
DANIEL J. How
596
?I!
1 i
-----
I
1,
1’ /;
k---___
10
0
Time
Fig. 3. Ion-pairing
purities illustrates
which
increased
reversed-phase detection, __
with
50
Imln)
HPLC of generator eluent. Conditions same as Fig. uv. detection); (*) pertechnetate, (a) artifacts.
generator
the chromatographic
40
30
20
age.
elution
Figure
6
of [““‘Tc]per-
technetate generator samples on days 1, 3, and 6. The starred peak indicates pertechnetate. 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 commercial MDP kit. The starred peaks in the trace, which contain y9mT~, have retention times corresponding to impurity peaks observed in the U.V. chromatogram of the generator eluent. This infers the 99mTc labeling of impurities. The presence of radiochemical impurities (e.g. “‘Cs, ‘“‘Ru, etc.) in the generator eluent has been
.
1: (-----) y-ray
reported.““,2’) This is believed to be an infrequent phenomenon and we have no evidence to suggest such occurred with the limited number of generators studied.
of Tc-MDP
Separation
mixtures
The MDP radiopharmaceutical kits provided by three manufacturers were all prepared identically and aliquots were taken at timed intervals for chromatographic evaluation. All three manufacturers’ products resulted in mixtures of chromatographically separable 99mT~-MDP complexes, as illustrated in Fig. 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
l d!.l::
0
I
I
I
I
I
10
20
30
40
50
Time
_1 0
-
I
, 10
( mm)
Fig. 4. Ion-pairing reversed-phase HPLC of commercial normal saline used to elute generator: conditions same as Fig. I: U.V. detection at 254 nm: (e) artifacts.
-._l
_J_
I
I
I
20
30
40
min
Fig. 5. Ion-pairing prepared
reversed-phase HPLC of normal saline with certified A.C.S. sodium chloride in purified water; u.v. detection at 254 nm; (0) artifacts.
Reversed-phase
HPLC
597
of 9YmTc-MDP
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 a greater partitioning phase (300 A) enables accessibility for suspected oligomeric 9’mTc-MDP complexes. At extended reaction times more y9mTc-MDP components 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 anionexchange HPLC. The charges ranged from - 1.5 to -8.0 with partial molar volumes of 510-1600 diphosphonate mL/mol. (I’) Mixed metal technetium complexes containing tin have also been suggested by Van den Brand.@?’
Conclusion
, 0
uu , 10
The MDP labeling kits, provided by three manufacturers, all yielded multiple chromatographically Y9mTc-MDP complexes. Ion-pairing separable 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. I
I
I
I
20
30
40
so
min Acknowledgements-This work was supported in part by Public Health Service Grant RNM ROl-CA40110-1 award by the National Cancer Institute, Department of Health and Human Services.
Fig. 6. Ion-pairing reversed-phase HPLC of generator eluent; conditions same as Fig. 1: (*) pertechnetate; (0) artifacts; samples of generator eluent eluted on days; (a) = 1, (b) = 3, and (c) = 6; U.V. detection at 254 nm.
I
0 Fig. 7. Ion-pairing denotes impurities
U 10
I
I
20
30
1
40
I
50
reversed-phase HPLC of Tc(Sn)MDP preparation; conditions in saline which have been labeled with 99mTc; (-u.v. detection,
same as Fig. 1: (*) y-ray detection).
DANIEL J.
598
(a)
I
0
HOCH and THOMASC. PINKERTON
4
a
(b)
!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
0
I 8
I 16
I
I
24
30
J 0
I 8
I 24
I 16
I 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.
References 1. Davis M. A. and Jones A. G. Semin. Nucl. Med. 6, 19 (1976). 2. Eckelman W. S. and Levenson S. M. Int. J. ADDI. .. Radiar. Isot. 28, 67 (1977). 3. Eckelman W. and Richards P. J. J. Nucl. Med. 11. 761
(1970). 4. Rudd T. G., Allen D. R. and Smith F. D. J. Nucl. Med 20, 821 (1979). 5. Tofe A. ‘J. and Francis M. D. J. Nucl. Med. 15, 69
(1974). 6. Srivastava S. C. and Richards P. Vol. 1 (CRC Press, Florida, 1983). 7. Najafi A. and Hutchison N. J. Nucl. Med. 26, 524 (1985). 8. Zimmer A. M. and Pave1 D. G. J. Nucl. Med. 18. 1230 (1977). 9. Russell C. D. and Majerik J. E. Inc. J. Appl. Radiat. Isot. 30, 753 (1979).
10. Pinkerton Anal. Gem.
T. C., Heineman W. R. and Deutsch E. 52, 1106 (1980).
11. Tenabe S., Zodda J. P., Libson K. et al. Inf. J. Appl. Radia?. Isof. 34, 1585 (1983).
12. Tanabe S., Zodda J. P., Deutsch E. et al. Inr. J. Appl. Radial. Isot. 34, 1577 (1983).
13. Wilson G. M. and Pinkerton T. C. Anal. Chem. 57,246 (1985). 14. Mikelsons M. V. and Pinkerton T. C. Anal. Chem. In press. 15. Husak V. and Vlcek J. Inl. J. Appl. Radiat. Isot. 30, 165 (1979).
16. Deutsch
E., Heineman
W. R., Zodda J. P. et al.
Im. J. Appl. Radial. Isot. 33, 843 (1982). 17. Lawson B. L., Powell C. R. and Pinkerton T. C. J. Radioanal. Nucl. Chem. Left. 92, 71 (1985). 18. Pacer R. A. Int. J. Appl. Radiat. Isot. 34, 731 (1980). 19. Bovd R. E. and Sorbv P. J. Inf. J. ADuI. . . Radiat. Isot. 35,-993 (1984). 20. Molinkski V. J. Inf. J. Appl. Radiaf. Isof. 33,811 (1982). 21. Finck R. and Mattsson S. Inf. J. Appl. Radial. Isot. 3, 89 (1976). 22. Van der Brand J. A. G. M. Netherlands Energy
Research Foundation, 1977 ZG. Petten (NH), The Netherlands, October (1981).
1986
0883-2889/86 $3.00 + 0.00 Pergamon Journals Ltd
Reversed-Phase HPLC of 99mTc Methylene Diphosphonate Bone Imaging Kits with Quantification of Pertechnetate DANIEL Department
J. HOCH
of Chemistry.
and
Purdue
THOMAS
University.
C. PINKERTON
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 kits utilizing 99mT~ labeled diphosphonates as bone imaging agents are widely used in the field of diagnostic nuclear medicine.“,‘) These rddiopharmaceutical kits contain a lyophilized mixture of reductant and ligand. Commercial use of tin as the reductantJ3) and methylene diphosphonate (MDP) as the bone seeking ligand, is widely accepted.‘4) The labeling procedure is carried out by the addition of the eluent from a %Mo/~~“Tc generator to the kit. The generator eluent contains [99mTc]pertechnetate in normal saline. The addition of 99mTc0; to commer,:ial diphosphonate kits is shown to yield a mixture of :nroducts at both the “carrier added” level as well as !he “no carrier added” level.(s ‘) The general labeling ,.eaction can be written as follows. Commercial
99mTc0; + Sn + MDP -+99mTc(Sn)MDP
(mixture
of complexes)
Previously, separation of product mixtures had Teen obtained with paper or thin layer chromatog.aphy. (8,9)These low performance methods character.stically distinguish only three components; y9mTc labeled product, unreduced pertechnetate, and hydrolyzed reduced pertechnetate. These quality control procedures lack the resolution necessary to evaluate the chemical nature of either the y9mTc labeled compounds or the hydrolyzed reduced technetium. An alternative separation method is electrophoresis. This has been shown to yield up to four 593
different 99mTc-MDP complexes; however, the technique does not facilitate the isolation of separated components for further investigation.“) High resolution separations of labeled 99mTc diphosphonate mixtures have been achieved with anion-exchange HPLC, utilizing polystyrene divinylbenzene strong ion-exchange resins.“&‘4) In contrast to Tc-HEDP preparations, the Tc-MDP complexes yield low recoveries when chromatographed on strong anion-exchangers, thus contributing to nonreproducibility and decreased column life.‘6,“.‘“) In addition, strong anion-exchange resins are not suited for the determination of total technetium concentration because high affinity prevents the elution of TcO;. In view of this, it is necessary to use guard columns and back-flushing techniques to remove unreacted pertechnetate from anion-exchange columns in order to prevent on-column reactions when an active reductant, such as Sn2+, remains in the injected sample.(‘3’ The total technetium concentration in a generator eluent has been shown to vary with the age and history of a generator, as well as the time since previous elution. (“.w Since pertechnetate cannot be eluted, strong anion-exchange HPLC cannot be used to quantify the total amount of technetium (i.e. “Tc and ““Tc) in the generator eluent prior to labeling. Ideally. the chromatographic method should be suitable for both (i) the determination of total technetium concentration in a generator eluent and (ii) the separation of labeled 99mTc-MDP complexes in a resultant reaction mixture.
DANIEL J. HOCH and THOMAS
594
This work presents an ion-pairing reversed-phase high performances liquid chromatographic separation of tin reduced 99mTc-MDP complexes on a macroporous silica bonded reversed-phase column. The radiopharmaceutical kits are prepared at “no carrier added” levels by manufacturer recommended procedures. The improved separation of the Tc(Sn) MDP reaction, as well as the increased recovery afforded by the use of ion-pairing reversed-phase HPLC, enables the profiling of 99mTc-MDP preparations and isolation of individual Tc-MDP complexes for subsequent characterization. Is also allows for the quantification of pertechnetate and the determination of chemical purity in generator eluents prior to labeling.
C.
PINKERTON
radiochemical purity, Oak Ridge Laboratories, Oak Ridge, Tenn.). The resulting potassium pertechnetate was recrystallized twice to insure purity. Samples of 99mTc present in generator eluents were provided by a regional distributor of radiopharmaceutical products. The Tc-MDP bone imaging kits were obtained from several manufacturers. Procedures The bone imaging kits were reconstituted according to manufacturers instructions. In each case 20 mCi of 99mTc, in I mL of normal saline generator eluent, was added to the commercial kit. The sample was allowed to sit for 20min before an aliquot was removed for injection onto the chromatographic column.
Experimental Apparatus The chromatographic equipment included a ConstaMetric Model III (Laboratory Data Control) dual piston pump, a Rheodyne Model 7125 injection valve with a 100 PL loop. The analytical column consisted of a 250 mm L x 4 mm i.d. stainless steel column containing a macroporous C-18 reversedphase packing (Vydac), with particle diameter of IO pm and 3OOA pore size. Detection methods included the use of an aluminum housed NaI(TI) well scintillation crystal (Harshaw, type 75F8) to detect radiation from the 99mTc (140 KeV yray tllz = 6.0 h) upon elution from the HPLC column. An Ortec model 420A timing single channel analyzer, Harshaw NA-23 amplifier, and a Harshaw NR linear ratemeter comprised the y detection electronics. A fixed wavelength Altex model 153 u.v.-visible liquid chromatography detector was utilized in-line after the scintillation detector. The fixed wavelength detector was operated at 254nm. Materials The mobile phase consisted of HPLC grade sodium acetate (Fisher Scientific), anhydrous reagent grade ethyl acetate (MCB Reagents) and tetrabutylammonium hydroxide (Aldrich). All solutions were prepared with distilled, deionized water which had been purified by a mixed bed ion-exchanger and 0.2pm filter with circulation through an activated carbon bed in a Water-I unit (Gelman Sciences). Final concentrations of the mobile phase constituents were 1 x lO_lM sodium acetate, 2 x IO-‘M tetrabutylammonium hydroxide, and 3% (v/v) ethyl acetate. The pH was adjusted to 6.0 with acetic acid. In house normal saline samples were prepared with certified A.C.S. sodium chloride (Fisher Scientific) diluted in distilled, deionized water further purified by the Water-I. Samples of normal saline commonly employed to elute technetium generators were obtained from several manufacturers. Potassium pertechnetate was obtained by the metatheses of ammonium pertechnetate (99%
Results and Discussion Quantljication
of pertechnetate
in generator
eluents
Solutions of pure, recrystallized pertechnetate (99Tc0; ) were subjected to ion-pairing reversedphase HPLC to determine retention time and percent recovery. Detection was accomplished by the use of a fixed wavelength U.V. detector at 254 nm. Samples prepared in water yielded a single peak with a retention time of 10.5 min and a recovery of > 98%. Since the 99mTc obtained from the technetium generator is supplied in normal saline (0.9% NaCI), standards were also prepared in saline. Injection of these samples yielded a split peak for pertechnetate (Fig. I). Through the use of a photodiode array u.v.-visible spectrophotometric detector, full wavelength (20~-8OOnm) spectra were obtained during chromatographic elution. Both peaks exhibited spectra identical to that of pertechnetate. The peak splitting observed appeared to be a chromatographic artifact caused by the large concentration of sodium chloride present in the sample, which tie up available ion-pairing reagent. This overloading of the ionpairing reagent causes a portion of the pertechnetate band to initially move ahead on the column. This phenomenon has not been reported by previous investigators using ion-pairing reversed-phase for the of the peak separation of MDP kits. (6) Elimination splitting artifact was accomplished by an appropriate dilution of the pertechnetate sample in the mobile phase. The minimum dilution required was determined empirically to be a 2: 3 mixture of mobile phase to sample (Fig. I). All subsequent samples which contained saline were diluted in this manner. Utilizing the 254 nm fixed wavelength detector, calibration curves were constructed by measuring chromatographic peak heights as a function of total pertechnetate (i.e. 99T~04 and 99”T~04) concentration. Over the “no carrier added” concentration range of I x IO-’ M to 5 x 10m6M a linear response was observed with a correlation coefficient of 0.999. At higher “carrier added” pertechnetate
Reversed-phase HPLC of WmT~-MDP
595
_1: A ( b)
(a)
I
I
I
I
0
6
16
0
L
I
I
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 IO-) M sodium acetate, 10m3M TBAOH, pH 6; mobile phase. Mobile phase 3% ethyl acetate, 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 I x 10m4M. A concurrent method to quantify total pertechnetate concentration is liquid scintillation counting.“‘,‘*) This requires at least a 60 h delay (10 half-lives) to (ensure complete decay of 99mTc to 99Tc. The HPLC :method described herein utilizes optical detection to [quantify the total pertechnetate and therefore can be Iused immediately on samples containing 99mTc. The ,:oncentration of pertechnetate in generator eluent samples, was determined by both procedures. Results If the measured concentrations from both methods .tre compared graphically in Fig. 2. A linear correation coefficient of 0.973 was obtained between the ~nethods. Chemical impurities
cial normal saline used to elute the generator. The number and relative magnitude of the unknown components are similar to that of the generator eluent. Figure 5 shows a chromatographic separation of normal saline prepared with certified A.C.S. sodium chloride in distilled/deionized/carbon filtered water. The first two peaks are solvent front artifacts. No responses were detected beyond 10min. The samples of [99mTc]pertechnetate generator eluent were studied over the clinically useful lifetime of two separate generators from the same manufacturer. Both generators were eluted with the same saline and both indicated the presence of U.V. absorbing im-
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 ^r 1 peak, as well as a corresponding U.V. peak, were 4 identical to that of pertechnetate. The remaining U.V. I: absorbing components were attributed to chemical a I i npurities in the generator eluent. Deutsch et a/.,(‘61 ./ 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 I s’ udy the source of the impurities was found to be in I I 1 2 3 the normal saline used to elute the generator and not the plastic column lining. This is in agreement with LSC (FM) the findings of Boyd,“‘) who also reported the presFig. 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 commerSlope = 1.01; correlation coefficient = 0.973.
1 /-
and THOMAS C. PINKERTON
DANIEL J. How
596
?I!
1 i
-----
I
1,
1’ /;
k---___
10
0
Time
Fig. 3. Ion-pairing
purities illustrates
which
increased
reversed-phase detection, __
with
50
Imln)
HPLC of generator eluent. Conditions same as Fig. uv. detection); (*) pertechnetate, (a) artifacts.
generator
the chromatographic
40
30
20
age.
elution
Figure
6
of [““‘Tc]per-
technetate generator samples on days 1, 3, and 6. The starred peak indicates pertechnetate. 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 commercial MDP kit. The starred peaks in the trace, which contain y9mT~, have retention times corresponding to impurity peaks observed in the U.V. chromatogram of the generator eluent. This infers the 99mTc labeling of impurities. The presence of radiochemical impurities (e.g. “‘Cs, ‘“‘Ru, etc.) in the generator eluent has been
.
1: (-----) y-ray
reported.““,2’) This is believed to be an infrequent phenomenon and we have no evidence to suggest such occurred with the limited number of generators studied.
of Tc-MDP
Separation
mixtures
The MDP radiopharmaceutical kits provided by three manufacturers were all prepared identically and aliquots were taken at timed intervals for chromatographic evaluation. All three manufacturers’ products resulted in mixtures of chromatographically separable 99mT~-MDP complexes, as illustrated in Fig. 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
l d!.l::
0
I
I
I
I
I
10
20
30
40
50
Time
_1 0
-
I
, 10
( mm)
Fig. 4. Ion-pairing reversed-phase HPLC of commercial normal saline used to elute generator: conditions same as Fig. I: U.V. detection at 254 nm: (e) artifacts.
-._l
_J_
I
I
I
20
30
40
min
Fig. 5. Ion-pairing prepared
reversed-phase HPLC of normal saline with certified A.C.S. sodium chloride in purified water; u.v. detection at 254 nm; (0) artifacts.
Reversed-phase
HPLC
597
of 9YmTc-MDP
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 a greater partitioning phase (300 A) enables accessibility for suspected oligomeric 9’mTc-MDP complexes. At extended reaction times more y9mTc-MDP components 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 anionexchange HPLC. The charges ranged from - 1.5 to -8.0 with partial molar volumes of 510-1600 diphosphonate mL/mol. (I’) Mixed metal technetium complexes containing tin have also been suggested by Van den Brand.@?’
Conclusion
, 0
uu , 10
The MDP labeling kits, provided by three manufacturers, all yielded multiple chromatographically Y9mTc-MDP complexes. Ion-pairing separable 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. I
I
I
I
20
30
40
so
min Acknowledgements-This work was supported in part by Public Health Service Grant RNM ROl-CA40110-1 award by the National Cancer Institute, Department of Health and Human Services.
Fig. 6. Ion-pairing reversed-phase HPLC of generator eluent; conditions same as Fig. 1: (*) pertechnetate; (0) artifacts; samples of generator eluent eluted on days; (a) = 1, (b) = 3, and (c) = 6; U.V. detection at 254 nm.
I
0 Fig. 7. Ion-pairing denotes impurities
U 10
I
I
20
30
1
40
I
50
reversed-phase HPLC of Tc(Sn)MDP preparation; conditions in saline which have been labeled with 99mTc; (-u.v. detection,
same as Fig. 1: (*) y-ray detection).
DANIEL J.
598
(a)
I
0
HOCH and THOMASC. PINKERTON
4
a
(b)
!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
0
I 8
I 16
I
I
24
30
J 0
I 8
I 24
I 16
I 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.
References 1. Davis M. A. and Jones A. G. Semin. Nucl. Med. 6, 19 (1976). 2. Eckelman W. S. and Levenson S. M. Int. J. ADDI. .. Radiar. Isot. 28, 67 (1977). 3. Eckelman W. and Richards P. J. J. Nucl. Med. 11. 761
(1970). 4. Rudd T. G., Allen D. R. and Smith F. D. J. Nucl. Med 20, 821 (1979). 5. Tofe A. ‘J. and Francis M. D. J. Nucl. Med. 15, 69
(1974). 6. Srivastava S. C. and Richards P. Vol. 1 (CRC Press, Florida, 1983). 7. Najafi A. and Hutchison N. J. Nucl. Med. 26, 524 (1985). 8. Zimmer A. M. and Pave1 D. G. J. Nucl. Med. 18. 1230 (1977). 9. Russell C. D. and Majerik J. E. Inc. J. Appl. Radiat. Isot. 30, 753 (1979).
10. Pinkerton Anal. Gem.
T. C., Heineman W. R. and Deutsch E. 52, 1106 (1980).
11. Tenabe S., Zodda J. P., Libson K. et al. Inf. J. Appl. Radia?. Isof. 34, 1585 (1983).
12. Tanabe S., Zodda J. P., Deutsch E. et al. Inr. J. Appl. Radial. Isot. 34, 1577 (1983).
13. Wilson G. M. and Pinkerton T. C. Anal. Chem. 57,246 (1985). 14. Mikelsons M. V. and Pinkerton T. C. Anal. Chem. In press. 15. Husak V. and Vlcek J. Inl. J. Appl. Radiat. Isot. 30, 165 (1979).
16. Deutsch
E., Heineman
W. R., Zodda J. P. et al.
Im. J. Appl. Radial. Isot. 33, 843 (1982). 17. Lawson B. L., Powell C. R. and Pinkerton T. C. J. Radioanal. Nucl. Chem. Left. 92, 71 (1985). 18. Pacer R. A. Int. J. Appl. Radiat. Isot. 34, 731 (1980). 19. Bovd R. E. and Sorbv P. J. Inf. J. ADuI. . . Radiat. Isot. 35,-993 (1984). 20. Molinkski V. J. Inf. J. Appl. Radiaf. Isof. 33,811 (1982). 21. Finck R. and Mattsson S. Inf. J. Appl. Radial. Isot. 3, 89 (1976). 22. Van der Brand J. A. G. M. Netherlands Energy
Research Foundation, 1977 ZG. Petten (NH), The Netherlands, October (1981).