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Comparison of different chromatographic quality-control procedures to determine the radiochemical purity of common 99mTc-diagnostic agents
Appl.Radial.Isor.Vol. 38, No. 4, pp. 283-288, 1987 Inr. J. Radial.Appl.Instrum.PartA
0883-2889/87$3.00+ 0.00 Copyright 0 1987Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
Comparison of Different Chromatographic Quality-Control Procedures to Determine the Radiochemical Purity of Common ggmTc-Diagnostic Agents G. L. ZUCCHINI,’
M. MARINELLI,’ and P. GARUTII
R. POZZAT03
‘Laboratory of Radiochemistry, Department of Chemistry, University of Ferrara, 44100 Ferrara, Italy, ‘Department of Nuclear Medicine, Maggiore Hospital, Bologna, Italy and 3Department of Health and Medical Physics, Maggiore Hospital, Bologna, Italy. (Received 28 February 1986; in revised form 15 September 1986) Simple and rapid chromatographic systems were used to test the radiochemical purity of eleven 99mTc-diagnostic agents. Those systems were selected which gave no oxidation or other by-products. The tests were done with equipment normally available in Nuclear Medical Departments.
Materials and Methods tests were carried out in air and in a glove-box where air had been replaced by nitrogen, scrubbed with a potassium pyrogallate solution (15% w/v of pyrogallic acid in 50% NaOH) in order to remove residual oxygen and prevent oxidation. The 99mTc-diagnostic agents shown in Table 1 were subjected to the tests. Paper and instant thin-layer chromatography was used to check the radiochemical purity of the %Tc-agents. A 0.9% sodium chloride solution USP (saline), a solvent of high polarity, and analytical grade acetone, solvent of lower polarity were used. Owing to the possible presence of peroxides, the acetone was renewed after every test. In order to detect the possible presence of undefined species in saline-developed chromatograms of relatively unstable agents, such as pyrophosphate,“) the strips were also developed with ligand solutions at the same Chromatographic
Table 1. Abbreviations of the examined *Tc-agents
TOG; TC-MDP Tc-HSA Tc-GH Tc-HANC Tc-PPi Tc-DISIDA Tc-MAA Tc-DTPA Tc-SSC Tc-SFC
Vc-pertechnetate ion W”Tc-methylendiphosphonate *Tc-human serum albumin 99mTc-gluceptate %Tc-human albumin nanocolloid 99mTc-pyrophosphate 99”Tc-disofenin W”Tc-macroaggregated albumin %Tc-pentetate WmTc-stannous sulphur colloid 99mTc-stannous fluoride colloid
concentration as the one used in the preparation each specific %Tc-agent.
of
Chromatographic procedure
Strips of Whatman No. 1 chromatographic paper (Whatman Inc.) and Type SG “instant” thin-layer medium (ITLC-SG, Gelman Instrument Co.), were routinely prepared in 6 x 15 cm size. Preliminary tests showed that 4cm is the minimum distance between the origins (marked by spots) necessary to avoid overlapping of the two chromatographic developments. For the convenience of the operator, each strip was also marked by dyed spots. These colored spots, moving with the solvent, were used as a reference point of the solvent front (Sr). Samples were one drop (2 ~1L) of the r@r”Tc-eluateapplied with a micropipette (Eppendorf) at the left side and another of the tested g9mTc-agent at the right side of each chromatographic strip. Without drying, the strips were promptly placed in the nitrogen-purged glove-box, suspended marked-end down in cylindrical glass tanks (volume 1 L) and dipped into 50 mL (height: 0.5 cm) of the developing solvent, which had previously been nitrogen-purged in gas washing bottles, and the tanks were then tightly covered. Chromatograms were all carried out at room temperature. When the solvent front had just reached l-2 cm from the top, as indicated by the dye, the strips were removed from the tanks, the solvent front being marked immediately, and dried within a few minutes in a stream of cold 283
284
G. L. ZUCCHINI et
nitrogen, and stored in the glove-box. The S, values were dependent upon conditions such as temperature, quality of the support material and pre-equilibration of the solution. Because of these variables, R, values, defined as the migration distance of the sample component divided by the migration distance of the solvent front, are not always reproducible. For this reason, the main suspected radiochemical impurity, i.e. pertechnetate ion, was always chromatographed simultaneously in the same tank and on the same strip as the 99mTc-complex being examined. The chromatographic procedure in air was the same as the one previously described in the nitrogenpurged glove-box. Four chromatographic tests were performed on each agent with the chromatographic procedures used in air and under nitrogen. Counting procedure
The activity distribution in relation to the distance on the chromatogram is usually performed either by scanning with a radiochromatographic scanner or by counting cut-up portions of the chromatogram in a scintillation well counter. A radiochromatographic scanner is not always available in nuclear medicine laboratories, and the chief drawback of the second method is the time required to complete the test in contrast with routine nuclear medicine operating times, since chromatogram reading involves many manual procedures. In order to simplify and shorten the test, we decided to use, in constant geometry of chromatograms on a support placed under the collimator of a computer (Dycom 80-Elscint) assisted gamma-camera (Searle LFOV), a commonly used device in nuclear medicine departments, with a measuring time of 30s. Origins and solvent front were indicated with “Co markers. The effect of the distance between gamma camera collimator and radioactive source on activity measurements was investigated in order to assess the optimal counting conditions as follows: -2pL of 99mTc-eluate were put on Whatman No. I strip; -the source was counted IO times at different distances from the collimator. On the basis of the results obtained, shown in Fig. 1, a distance of 12cm was used to measure the activity of the chromatograms. Each chromatographic
fraction activity was deter-
Table 2. R: (%)t values of vc-eluate Agent *“TcO;
al.
4.5
“0 x E m ‘0 :I
4.0
l.
3.5b *4t1 0
’
6
’
7
’
6
’
9
’
10
’
11
’
12
’
’
13 14
cm Fig. 1. Effect of the distance “source-collimator” on the measured activity. mined with the aid of a square ROI (region of interest), 5 x 5 pixel, running along the strip step and expressed as a percentage of the total activity detected on the strip. All the counts were corrected for the 99mT~ decay and recorded as net counts per minute (cpm), background subtracted. The proposed counting method allowed us to evaluate activities of about 5 pCi with the precision of 1% on both supports (Whatman No. 1 paper and ITLC-SG). Statistics
A set of statistical tests (x2, F and t-test) was carried out to assess whether or not the experimental procedure previously described causes significant “errors” in relation to the variability connected with the radioactive decay. Gamma camera. A source, prepared by putting 2 p L of 99mTc-eluate on a Whatman No. I strip, was counted 10 times in a fixed position. Then, the same source, randomly positioned, was counted 10 times again. The x2 test showed that neither the instrument itself nor the random positioning of the source causes statistically significant errors (P = 0.05). Micropipette and supports. Two series of 10 samples on Whatman No. 1 paper and ITLC-SG strips, each one prepared by adding 2 PL of 99mTc-eluate, were examined. Since the results of the x2 test was negative at the 5% significance level (unlike Poisson counting distri-
with different solvents in air and in nitrogen with Whatman No. 1 paper and ITLC-SG
No. of replicates
Solvent
Whatman No. 1 paper
ITLC-SG
in air
40 40 12
Saline Acetone Ligand
0.7-0.9 (9~100%) 0.9-I .o (9&99%) 0.7-1.0 (88-96%)
0.9-1.0(91-100%) 0.9-1.0(91-100%) 1.0 (97-100%)
in N,
32 32 28
Saline Acetone Ligand
0.74.9(93-100%) 0.9-1.0 (92-100%) 0.7-1.0 (89-98%)
0.9-I .o (9&100%) 0.9-1.0 (9&100%) 1.O(92-100%)
lRf range of wmTc-eluate obtained by adopted chromatographic t(X)
5
methods. percentage range of detected 99mTc-elutate related to R, range.
Comparison of chromatographic quality-control procedures
bution) for both supports, sample standard deviation (S) must be associated with both average values. The t-test showed, at a 5% significance level, that the two average values were statistically equal. Consequently, it makes no difference which support is used to measure the activity of solution samples. The adopted 5% uncertainty level is generally acceptable for analytical test results.@)
Results and Discussion Pertechnetate The quantitative results of comparative investigations in air and in nitrogen by chromatography on Whatman No. 1 paper and ITLC-SG, using three solvent systems, are given in Table 2. As data show, pertechnetate migrates close to the acetone solvent front (R,: 0.9-1.0) on both supports; with aqueous solvents the behaviour is qualitatively the same, but quantitatively different, regardless of the type of support. On paper the migration of the pertechnetate is on average lower, both with the saline (R,: 0.749) and with the ligand solution (R,: 0.7-1.0) whereas with ITLC-SG the migration is the same as that obtained with acetone (R,: 0.9-1.0) for both the solvents. Technetium-99m agents The complete quantitative results for radiochemical purity of specific 99mTc-agents obtained with the above-mentioned chromatographic systems are presented in Table 3. Having experimentally defined the R, values for the pertechnetate (Table 2), that for “reducedhydrolyzed ” 99mTcbeing well known (R, = 0) since it is highly insoluble and so remains as radiocolloid (TcO,) at the origin, the *Tc-agent R,s (bound 99mTc)were calculated from the chromatograms. The percentage of activity relative to each of the three species detected on the chromatograms was indicated in brackets beside the corresponding RI values. Any presence of activity due to undefined species labelled with 99mTc was also expressed in percentage and reported in brackets under “Trial”. The variables to be considered in the interpretation of the experimental values obtained are: physicalchemical characteristics of the wmTc-agents examined, atmosphere of the tests, type of support, nature of the mobile phase, and development time. One must bear in mind that generally it is not possible to attribute certain results to the effect of a single variable. Comparing the data obtained by working in air and nitrogen, the latter seems to be preferable for Tc-MDP and more so for Tc-GH, whereas this was not so for the other agents. The chromatographic tests carried out on ITLCSG showed development times which were clearly shorter than the corresponding ones obtained on paper.
285
This characteristic makes ITLC-SG’ preferable in that it considerably reduces the time needed to perform the entire chromatographic procedure, thus reducing the amount of dissociation and reoxidation of reduced 99mTcto pertechnetate that can occur in the development procedure.‘” For example, the best result obtained with Tc-MDP by using ITLC-SG (7 min) compared with that on paper (18 min) may be attributed to the more rapid development, combined with the effect of the ligand solution. Even more evident was the positive influence of the short times with Tc-HSA and Tc-GH. By using ITLC-SG, it was possible to examine Tc-HSA with all three solvents both in air and nitrogen, and obtained positive results as far as the radiochemical purity was concerned, whereas with Tc-SG the best results of the tests with ITLC-SG seem to be due also to the fact that they were carried out in nitrogen. Among the solvents, acetone provided positive results in practically all cases, except for Tc-PPi and Tc-DISIDA. The aqueous solvents (saline, ligand solution), on the other hand, produced discordant results. Of the two, the ligand solution worked better especially when used in combination with ITLC-SG. Finally, with Tc-PPi and Tc-DISIDA no acceptable result was obtained. The high rate of radiochemical impurity systematically observed in all the tests may be ascribed, in our opinion, to the physico-chemical characteristics of these 99mTc-agents. Concerning the undefined species, the continuous distribution (trail) of activity observed between the origin and pertechnetate is probably indicative of various oxidized or complexed states of technetium, and their presence seems to be due, in order of importance, to the aqueous solvents (mainly saline), the nature of the support (mainly paper) and, finally to the chosen atmosphere (mainly air). This general pattern of the influence level of the experimental variables which contribute to the undefined species production was confirmed both in the 99mTc-agent tests which show positive and negative results, and in the tests of those agents (Tc-PPi, Tc-DISIDA) whose results were all negative. The most significant factor in 99mTc-chromatography is a complete understanding of each system’s function and limitation. In Table 3, therefore, the positive tests regarding every 99mTc-agent examined are clearly indicated for those interested in this field of investigation. So it is evident that in only one case the three species of interest (free, reduced-hydrolyzed, bound 99mTc) may be detected by only one of the chromatographic systems used (Tc-DTPA: Whatman No. 1 paper/saline in air) in that only reduced-hydrolyzed 99mTcremains at the origin, while pertechnetate and 99mTc-agent move with different Rf values. Thus, this system provides a complete picture of the radiochemical composition of 99mTc-DTPA. In some other cases (Tc-MDP, Tc-GH, Tc-DTPA), the same results may be obtained only by using a combination of two distinct chromatographic systems. It
**(66) ~(86)
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(P) 0 CP.“) 0 (00
(z) 0 W”) 0 (0) 0
(9) L’O (0) 6’0 (51) L’O
CP.U)0’1 (010’1 (sz) L’O
(‘p’“)o’I (O)o’i CT”) L’O
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CPWO CPW0 (T4 0
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(0) (0) (0) ~(96) r&6) *r&6) 0 0 0
'.P;%:;
(0) (0) (0)
l*w ~(86) r&6) 0 0
0 0
(0) (0) (0) ~(96) **(%) 40000
01 L 8
i.(P) i.(P) (0)
cF4 0 CT.90 CPN 0 (z)o CP-) 0 (00 ZI ZI ZI
*?a(%) 0’ I 486) 0 (s-9 0’ 1
(z) 6’0
i(E) (0) i.(W)
HtY3.l
VSH-X
I-
I..
21 12 18
19 II
19 II
II
0 (n.d.) 0 (n.d.)
0.7 (1)
1.o(0) 0.9 (0) 0.9 (0) 1.o (0)
9 IO IO
0.7 (0)
0.9 (3) 0.9 (0) 0.8 (2)
12 11
10 10 10
1.o(0)
0.7 (0) 0.9 (0)
II II 12 12
0 (0) 0 (n.d.)
0.7 (0) 0.9 (0)
12 11
0 (n.d.) 0 (n.d.) 0 (n.d.)
0 (n.d.) 0 (n.d.)
0 (n.d.) O(n.d.) 0 (n.d.)
0 (n.d.) 0 (n.d.)
0 (2) 008) O(I)
0.8 (n.d.) 0.9 (n.d.) 0.9 (n.d.)
11 11 11
0 (3) 0 (23)
O(11) 0 (38) O(8)
0 (n.d.)
ow
0.7 (n.d.) 0.9 (n.d.)
0.7 (n.d.) I .O(n.d.) 0.7 (10)
0.7 (n.d.) 0.9 (26)
11 II
II 10 11
11
0 (98)” 0 @I)** 0 (98);.
0 (98)** 0 (99)**
0 (80) 0 (99)‘; 0 (86)
0 (76) 0 (98)‘*
0.9(100)*** o(loo)*’
0 (loo)** o(lOO)**
0.8 (52) 0.9 (61) 0.9 (80)
0.7 (50) 0.9 (47)
0.7 (57) 1.0(H) 0.9 (54)
0.7 (38) 0 (64)
w
(2)? (0)
(2)? (0)
(17)? (0) (12)?
(23)? (2)?
(0) (0)
(0) (0)
WY (21)? (19)?
(47)? (30)?
(32)? (II)? (2X)?
(52)? (IO)?
._._.”
-
- ---._.--
--.
.
._-.
-_.---
* Detined as the migration distance of the sampk component divided by the migration distance of the solvent front. t Percentages of free, hydrolyscd-reduced and bound *Tc in 9R”Tc-diagnostic agent preparations. $ Refers to a solution of the specific @and at the same concentration as that used to prepare the %Tc-agent considered. (n.d.) Not definabk percentage owing to the coincidence of the & species and that of the labelled one. (%)? Percentage of indefinable species (artifacts). ** Detection of two specks (TcO; and labelkd or TcO, and labelled). *** Detection of three species (TcO; , TcO, and labelled).
Saline Acetone Ligand
Saline Acetone
Tc-SFC
4 4 4
Saline Acetolle Ligand
4 4 4
in Nz
in N2
19 13
Saline Acetone
4 4
in air
Tc-ssc
4 4
21 12 16
Saline Acetone
4 4
in air
Tc-DTPA
in air
19 13
Saline Acetone
4 4
in air
TC-MAA
18 12 20
Saline Acetone Ligand
I8 11
4 4 4
Tc-DISIDA
in Nz
18 12 17
Saline Acetone Ligand
Saline Acetone
4 4 4
in N,
18 II
Saline Acetone
4 4
4 4
in air
in air
Tc-PPi
11 II 12 I2 12 12 II
13 5 9 7 6 13 5 7
-_..-
11 II
7 6
II
12 12
12 12
11 11 12
10 6
10 6
13 6 9
12 10
12 II 12
12 6 6 7 5
12 II
7 5
0 (n.d.) 0 (n.d.)
1.o(0)
0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.)
I .o(0) 1.o(2) I .o(0) 1.o(0) I .o(0)
._-
0.9 (0) 1.o (0)
0.9 (0)
1.o (2)
0.9 (0)
0 (0) 0 (n.d.)
1.O(n.d.) 0.9 (1)
0.9 (0)
0 (7) 0 (9) 0 (0)
0 (0) 0 (5)
0 (5) 0 (9) O(l)
0 (n.d.)
ow
0.9 (n.d.) 0.9 (7)? 1.o (3)?
1.O(n.d.) 0.9 (n.d.)
1.O(8) 1.9(8) 1.O(n.d.)
I .O(n.d.) 0.8 (43)
I--cl_.lil.._^“,._“-__
o(lW)**
..__...
0 (99)** 0 (99).f
0 (%).f 0 (se)**
0 (86) 0 (98)*’ 0 (92)
0 (85) 0 (95)**
0 (98)**
1.o(95)**
o(lOO)** 0 (97)”
0.9 (68) 0.8 (73) 0.9 (72)
0.8 (39) 0.9 (66)
0.9 (61) 0.8 (65) 1.O(80)
0 P-9
I .o(46)
-
(0)
(0) (0)
(4)? (2Y
(12)? (2)? (6)?
(15)? (5)?
(5)? (I)?
(0) (3Y
(25)? (21)? (ZS)?
(59)? (29)?
(26)? (18)? (19)?
(17)?
W)?
-.
-
_
G.
288
L.
ZUCCHINI et al.
is
evident from the results obtained that the combination works only if the 99mTc-agent examined migrates in one of the two systems used. If this condition is not fulfilled (Tc-HSA, Tc-HANC, Tc-MAA, Tc-SSC, Tc-SFC), the number of detectable species is limited to two (free and presumably bound %“Tc). Finally, in two cases (Tc-PPi, Tc-DISIDA) none of the chromatographic systems used provided satisfactory results as far as the radiochemical purity was concerned.
Conclusions The chromatographic systems used were generally found to be suitable for the determination of the radiochemical purity of the 99mTc-diagnostic agents examined. The better results obtained by one system
as compared to the others depend mainly on the type of support and solvent used, whereas there was little or no effect on the results if the experiments were done in air or nitrogen. These chromatographic systems did not provide satisfactory results for Tc-PPi and Tc-DISIDA respectively, due to the physico-chemical properties of the two agents.
References 1. Clarke M. J. and Fackler P. N. Structure
and Bondinn
50, 57 (1982).
Laitinen H. A. Chemical Analysis. An Advanced Text 2’ and Reference, p. 547. (McGraw-Hill, New York, 1968). 3, Zimmer A. M. and Pave1D. G. J. Nucl. Med. 18, 1230 (1977).
0883-2889/87$3.00+ 0.00 Copyright 0 1987Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
Comparison of Different Chromatographic Quality-Control Procedures to Determine the Radiochemical Purity of Common ggmTc-Diagnostic Agents G. L. ZUCCHINI,’
M. MARINELLI,’ and P. GARUTII
R. POZZAT03
‘Laboratory of Radiochemistry, Department of Chemistry, University of Ferrara, 44100 Ferrara, Italy, ‘Department of Nuclear Medicine, Maggiore Hospital, Bologna, Italy and 3Department of Health and Medical Physics, Maggiore Hospital, Bologna, Italy. (Received 28 February 1986; in revised form 15 September 1986) Simple and rapid chromatographic systems were used to test the radiochemical purity of eleven 99mTc-diagnostic agents. Those systems were selected which gave no oxidation or other by-products. The tests were done with equipment normally available in Nuclear Medical Departments.
Materials and Methods tests were carried out in air and in a glove-box where air had been replaced by nitrogen, scrubbed with a potassium pyrogallate solution (15% w/v of pyrogallic acid in 50% NaOH) in order to remove residual oxygen and prevent oxidation. The 99mTc-diagnostic agents shown in Table 1 were subjected to the tests. Paper and instant thin-layer chromatography was used to check the radiochemical purity of the %Tc-agents. A 0.9% sodium chloride solution USP (saline), a solvent of high polarity, and analytical grade acetone, solvent of lower polarity were used. Owing to the possible presence of peroxides, the acetone was renewed after every test. In order to detect the possible presence of undefined species in saline-developed chromatograms of relatively unstable agents, such as pyrophosphate,“) the strips were also developed with ligand solutions at the same Chromatographic
Table 1. Abbreviations of the examined *Tc-agents
TOG; TC-MDP Tc-HSA Tc-GH Tc-HANC Tc-PPi Tc-DISIDA Tc-MAA Tc-DTPA Tc-SSC Tc-SFC
Vc-pertechnetate ion W”Tc-methylendiphosphonate *Tc-human serum albumin 99mTc-gluceptate %Tc-human albumin nanocolloid 99mTc-pyrophosphate 99”Tc-disofenin W”Tc-macroaggregated albumin %Tc-pentetate WmTc-stannous sulphur colloid 99mTc-stannous fluoride colloid
concentration as the one used in the preparation each specific %Tc-agent.
of
Chromatographic procedure
Strips of Whatman No. 1 chromatographic paper (Whatman Inc.) and Type SG “instant” thin-layer medium (ITLC-SG, Gelman Instrument Co.), were routinely prepared in 6 x 15 cm size. Preliminary tests showed that 4cm is the minimum distance between the origins (marked by spots) necessary to avoid overlapping of the two chromatographic developments. For the convenience of the operator, each strip was also marked by dyed spots. These colored spots, moving with the solvent, were used as a reference point of the solvent front (Sr). Samples were one drop (2 ~1L) of the r@r”Tc-eluateapplied with a micropipette (Eppendorf) at the left side and another of the tested g9mTc-agent at the right side of each chromatographic strip. Without drying, the strips were promptly placed in the nitrogen-purged glove-box, suspended marked-end down in cylindrical glass tanks (volume 1 L) and dipped into 50 mL (height: 0.5 cm) of the developing solvent, which had previously been nitrogen-purged in gas washing bottles, and the tanks were then tightly covered. Chromatograms were all carried out at room temperature. When the solvent front had just reached l-2 cm from the top, as indicated by the dye, the strips were removed from the tanks, the solvent front being marked immediately, and dried within a few minutes in a stream of cold 283
284
G. L. ZUCCHINI et
nitrogen, and stored in the glove-box. The S, values were dependent upon conditions such as temperature, quality of the support material and pre-equilibration of the solution. Because of these variables, R, values, defined as the migration distance of the sample component divided by the migration distance of the solvent front, are not always reproducible. For this reason, the main suspected radiochemical impurity, i.e. pertechnetate ion, was always chromatographed simultaneously in the same tank and on the same strip as the 99mTc-complex being examined. The chromatographic procedure in air was the same as the one previously described in the nitrogenpurged glove-box. Four chromatographic tests were performed on each agent with the chromatographic procedures used in air and under nitrogen. Counting procedure
The activity distribution in relation to the distance on the chromatogram is usually performed either by scanning with a radiochromatographic scanner or by counting cut-up portions of the chromatogram in a scintillation well counter. A radiochromatographic scanner is not always available in nuclear medicine laboratories, and the chief drawback of the second method is the time required to complete the test in contrast with routine nuclear medicine operating times, since chromatogram reading involves many manual procedures. In order to simplify and shorten the test, we decided to use, in constant geometry of chromatograms on a support placed under the collimator of a computer (Dycom 80-Elscint) assisted gamma-camera (Searle LFOV), a commonly used device in nuclear medicine departments, with a measuring time of 30s. Origins and solvent front were indicated with “Co markers. The effect of the distance between gamma camera collimator and radioactive source on activity measurements was investigated in order to assess the optimal counting conditions as follows: -2pL of 99mTc-eluate were put on Whatman No. I strip; -the source was counted IO times at different distances from the collimator. On the basis of the results obtained, shown in Fig. 1, a distance of 12cm was used to measure the activity of the chromatograms. Each chromatographic
fraction activity was deter-
Table 2. R: (%)t values of vc-eluate Agent *“TcO;
al.
4.5
“0 x E m ‘0 :I
4.0
l.
3.5b *4t1 0
’
6
’
7
’
6
’
9
’
10
’
11
’
12
’
’
13 14
cm Fig. 1. Effect of the distance “source-collimator” on the measured activity. mined with the aid of a square ROI (region of interest), 5 x 5 pixel, running along the strip step and expressed as a percentage of the total activity detected on the strip. All the counts were corrected for the 99mT~ decay and recorded as net counts per minute (cpm), background subtracted. The proposed counting method allowed us to evaluate activities of about 5 pCi with the precision of 1% on both supports (Whatman No. 1 paper and ITLC-SG). Statistics
A set of statistical tests (x2, F and t-test) was carried out to assess whether or not the experimental procedure previously described causes significant “errors” in relation to the variability connected with the radioactive decay. Gamma camera. A source, prepared by putting 2 p L of 99mTc-eluate on a Whatman No. I strip, was counted 10 times in a fixed position. Then, the same source, randomly positioned, was counted 10 times again. The x2 test showed that neither the instrument itself nor the random positioning of the source causes statistically significant errors (P = 0.05). Micropipette and supports. Two series of 10 samples on Whatman No. 1 paper and ITLC-SG strips, each one prepared by adding 2 PL of 99mTc-eluate, were examined. Since the results of the x2 test was negative at the 5% significance level (unlike Poisson counting distri-
with different solvents in air and in nitrogen with Whatman No. 1 paper and ITLC-SG
No. of replicates
Solvent
Whatman No. 1 paper
ITLC-SG
in air
40 40 12
Saline Acetone Ligand
0.7-0.9 (9~100%) 0.9-I .o (9&99%) 0.7-1.0 (88-96%)
0.9-1.0(91-100%) 0.9-1.0(91-100%) 1.0 (97-100%)
in N,
32 32 28
Saline Acetone Ligand
0.74.9(93-100%) 0.9-1.0 (92-100%) 0.7-1.0 (89-98%)
0.9-I .o (9&100%) 0.9-1.0 (9&100%) 1.O(92-100%)
lRf range of wmTc-eluate obtained by adopted chromatographic t(X)
5
methods. percentage range of detected 99mTc-elutate related to R, range.
Comparison of chromatographic quality-control procedures
bution) for both supports, sample standard deviation (S) must be associated with both average values. The t-test showed, at a 5% significance level, that the two average values were statistically equal. Consequently, it makes no difference which support is used to measure the activity of solution samples. The adopted 5% uncertainty level is generally acceptable for analytical test results.@)
Results and Discussion Pertechnetate The quantitative results of comparative investigations in air and in nitrogen by chromatography on Whatman No. 1 paper and ITLC-SG, using three solvent systems, are given in Table 2. As data show, pertechnetate migrates close to the acetone solvent front (R,: 0.9-1.0) on both supports; with aqueous solvents the behaviour is qualitatively the same, but quantitatively different, regardless of the type of support. On paper the migration of the pertechnetate is on average lower, both with the saline (R,: 0.749) and with the ligand solution (R,: 0.7-1.0) whereas with ITLC-SG the migration is the same as that obtained with acetone (R,: 0.9-1.0) for both the solvents. Technetium-99m agents The complete quantitative results for radiochemical purity of specific 99mTc-agents obtained with the above-mentioned chromatographic systems are presented in Table 3. Having experimentally defined the R, values for the pertechnetate (Table 2), that for “reducedhydrolyzed ” 99mTcbeing well known (R, = 0) since it is highly insoluble and so remains as radiocolloid (TcO,) at the origin, the *Tc-agent R,s (bound 99mTc)were calculated from the chromatograms. The percentage of activity relative to each of the three species detected on the chromatograms was indicated in brackets beside the corresponding RI values. Any presence of activity due to undefined species labelled with 99mTc was also expressed in percentage and reported in brackets under “Trial”. The variables to be considered in the interpretation of the experimental values obtained are: physicalchemical characteristics of the wmTc-agents examined, atmosphere of the tests, type of support, nature of the mobile phase, and development time. One must bear in mind that generally it is not possible to attribute certain results to the effect of a single variable. Comparing the data obtained by working in air and nitrogen, the latter seems to be preferable for Tc-MDP and more so for Tc-GH, whereas this was not so for the other agents. The chromatographic tests carried out on ITLCSG showed development times which were clearly shorter than the corresponding ones obtained on paper.
285
This characteristic makes ITLC-SG’ preferable in that it considerably reduces the time needed to perform the entire chromatographic procedure, thus reducing the amount of dissociation and reoxidation of reduced 99mTcto pertechnetate that can occur in the development procedure.‘” For example, the best result obtained with Tc-MDP by using ITLC-SG (7 min) compared with that on paper (18 min) may be attributed to the more rapid development, combined with the effect of the ligand solution. Even more evident was the positive influence of the short times with Tc-HSA and Tc-GH. By using ITLC-SG, it was possible to examine Tc-HSA with all three solvents both in air and nitrogen, and obtained positive results as far as the radiochemical purity was concerned, whereas with Tc-SG the best results of the tests with ITLC-SG seem to be due also to the fact that they were carried out in nitrogen. Among the solvents, acetone provided positive results in practically all cases, except for Tc-PPi and Tc-DISIDA. The aqueous solvents (saline, ligand solution), on the other hand, produced discordant results. Of the two, the ligand solution worked better especially when used in combination with ITLC-SG. Finally, with Tc-PPi and Tc-DISIDA no acceptable result was obtained. The high rate of radiochemical impurity systematically observed in all the tests may be ascribed, in our opinion, to the physico-chemical characteristics of these 99mTc-agents. Concerning the undefined species, the continuous distribution (trail) of activity observed between the origin and pertechnetate is probably indicative of various oxidized or complexed states of technetium, and their presence seems to be due, in order of importance, to the aqueous solvents (mainly saline), the nature of the support (mainly paper) and, finally to the chosen atmosphere (mainly air). This general pattern of the influence level of the experimental variables which contribute to the undefined species production was confirmed both in the 99mTc-agent tests which show positive and negative results, and in the tests of those agents (Tc-PPi, Tc-DISIDA) whose results were all negative. The most significant factor in 99mTc-chromatography is a complete understanding of each system’s function and limitation. In Table 3, therefore, the positive tests regarding every 99mTc-agent examined are clearly indicated for those interested in this field of investigation. So it is evident that in only one case the three species of interest (free, reduced-hydrolyzed, bound 99mTc) may be detected by only one of the chromatographic systems used (Tc-DTPA: Whatman No. 1 paper/saline in air) in that only reduced-hydrolyzed 99mTcremains at the origin, while pertechnetate and 99mTc-agent move with different Rf values. Thus, this system provides a complete picture of the radiochemical composition of 99mTc-DTPA. In some other cases (Tc-MDP, Tc-GH, Tc-DTPA), the same results may be obtained only by using a combination of two distinct chromatographic systems. It
**(66) ~(86)
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cF4 0 CT.90 CPN 0 (z)o CP-) 0 (00 ZI ZI ZI
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i(E) (0) i.(W)
HtY3.l
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21 12 18
19 II
19 II
II
0 (n.d.) 0 (n.d.)
0.7 (1)
1.o(0) 0.9 (0) 0.9 (0) 1.o (0)
9 IO IO
0.7 (0)
0.9 (3) 0.9 (0) 0.8 (2)
12 11
10 10 10
1.o(0)
0.7 (0) 0.9 (0)
II II 12 12
0 (0) 0 (n.d.)
0.7 (0) 0.9 (0)
12 11
0 (n.d.) 0 (n.d.) 0 (n.d.)
0 (n.d.) 0 (n.d.)
0 (n.d.) O(n.d.) 0 (n.d.)
0 (n.d.) 0 (n.d.)
0 (2) 008) O(I)
0.8 (n.d.) 0.9 (n.d.) 0.9 (n.d.)
11 11 11
0 (3) 0 (23)
O(11) 0 (38) O(8)
0 (n.d.)
ow
0.7 (n.d.) 0.9 (n.d.)
0.7 (n.d.) I .O(n.d.) 0.7 (10)
0.7 (n.d.) 0.9 (26)
11 II
II 10 11
11
0 (98)” 0 @I)** 0 (98);.
0 (98)** 0 (99)**
0 (80) 0 (99)‘; 0 (86)
0 (76) 0 (98)‘*
0.9(100)*** o(loo)*’
0 (loo)** o(lOO)**
0.8 (52) 0.9 (61) 0.9 (80)
0.7 (50) 0.9 (47)
0.7 (57) 1.0(H) 0.9 (54)
0.7 (38) 0 (64)
w
(2)? (0)
(2)? (0)
(17)? (0) (12)?
(23)? (2)?
(0) (0)
(0) (0)
WY (21)? (19)?
(47)? (30)?
(32)? (II)? (2X)?
(52)? (IO)?
._._.”
-
- ---._.--
--.
.
._-.
-_.---
* Detined as the migration distance of the sampk component divided by the migration distance of the solvent front. t Percentages of free, hydrolyscd-reduced and bound *Tc in 9R”Tc-diagnostic agent preparations. $ Refers to a solution of the specific @and at the same concentration as that used to prepare the %Tc-agent considered. (n.d.) Not definabk percentage owing to the coincidence of the & species and that of the labelled one. (%)? Percentage of indefinable species (artifacts). ** Detection of two specks (TcO; and labelkd or TcO, and labelled). *** Detection of three species (TcO; , TcO, and labelled).
Saline Acetone Ligand
Saline Acetone
Tc-SFC
4 4 4
Saline Acetolle Ligand
4 4 4
in Nz
in N2
19 13
Saline Acetone
4 4
in air
Tc-ssc
4 4
21 12 16
Saline Acetone
4 4
in air
Tc-DTPA
in air
19 13
Saline Acetone
4 4
in air
TC-MAA
18 12 20
Saline Acetone Ligand
I8 11
4 4 4
Tc-DISIDA
in Nz
18 12 17
Saline Acetone Ligand
Saline Acetone
4 4 4
in N,
18 II
Saline Acetone
4 4
4 4
in air
in air
Tc-PPi
11 II 12 I2 12 12 II
13 5 9 7 6 13 5 7
-_..-
11 II
7 6
II
12 12
12 12
11 11 12
10 6
10 6
13 6 9
12 10
12 II 12
12 6 6 7 5
12 II
7 5
0 (n.d.) 0 (n.d.)
1.o(0)
0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.) 0 (n.d.)
I .o(0) 1.o(2) I .o(0) 1.o(0) I .o(0)
._-
0.9 (0) 1.o (0)
0.9 (0)
1.o (2)
0.9 (0)
0 (0) 0 (n.d.)
1.O(n.d.) 0.9 (1)
0.9 (0)
0 (7) 0 (9) 0 (0)
0 (0) 0 (5)
0 (5) 0 (9) O(l)
0 (n.d.)
ow
0.9 (n.d.) 0.9 (7)? 1.o (3)?
1.O(n.d.) 0.9 (n.d.)
1.O(8) 1.9(8) 1.O(n.d.)
I .O(n.d.) 0.8 (43)
I--cl_.lil.._^“,._“-__
o(lW)**
..__...
0 (99)** 0 (99).f
0 (%).f 0 (se)**
0 (86) 0 (98)*’ 0 (92)
0 (85) 0 (95)**
0 (98)**
1.o(95)**
o(lOO)** 0 (97)”
0.9 (68) 0.8 (73) 0.9 (72)
0.8 (39) 0.9 (66)
0.9 (61) 0.8 (65) 1.O(80)
0 P-9
I .o(46)
-
(0)
(0) (0)
(4)? (2Y
(12)? (2)? (6)?
(15)? (5)?
(5)? (I)?
(0) (3Y
(25)? (21)? (ZS)?
(59)? (29)?
(26)? (18)? (19)?
(17)?
W)?
-.
-
_
G.
288
L.
ZUCCHINI et al.
is
evident from the results obtained that the combination works only if the 99mTc-agent examined migrates in one of the two systems used. If this condition is not fulfilled (Tc-HSA, Tc-HANC, Tc-MAA, Tc-SSC, Tc-SFC), the number of detectable species is limited to two (free and presumably bound %“Tc). Finally, in two cases (Tc-PPi, Tc-DISIDA) none of the chromatographic systems used provided satisfactory results as far as the radiochemical purity was concerned.
Conclusions The chromatographic systems used were generally found to be suitable for the determination of the radiochemical purity of the 99mTc-diagnostic agents examined. The better results obtained by one system
as compared to the others depend mainly on the type of support and solvent used, whereas there was little or no effect on the results if the experiments were done in air or nitrogen. These chromatographic systems did not provide satisfactory results for Tc-PPi and Tc-DISIDA respectively, due to the physico-chemical properties of the two agents.
References 1. Clarke M. J. and Fackler P. N. Structure
and Bondinn
50, 57 (1982).
Laitinen H. A. Chemical Analysis. An Advanced Text 2’ and Reference, p. 547. (McGraw-Hill, New York, 1968). 3, Zimmer A. M. and Pave1D. G. J. Nucl. Med. 18, 1230 (1977).