Influence of the Generator in-Growth Time on the Final Radiochemical Purity and Stability of 99 mTc Radiopharmaceuticals

1 Dipartimento di Morfologia, Chirurgia e Medicina Sperimentale, Sezione di Diagnostica per Immagini, Università di Ferrara and INFN, Sezione di Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy 2 Dipartimento di Fisica e Scienze della Terra, Università di Ferrara and INFN, Sezione di Ferrara, Via Saragat 1, 44122 Ferrara, Italy 3 INFN, Laboratori Nazionali di Legnaro (LNL), Via dell’Università 2, 35020 Legnaro, Italy


Introduction
99m Tc, with its peculiar physical-chemical properties, still continues to be the most important radionuclide used in diagnostic nuclear medical procedures.In particular, the developments of technetium chemistry have opened new perspectives in the field of diagnostic imaging [1].More than 80% of the radiopharmaceuticals are currently labeled with this radionuclide [1] by reconstitution with sodium pertechnetate [2][3][4] [Na 99m TcO 4 ] commercial kits containing in lyophilized form the various reagents required for the preparation of each radiopharmaceutical.Its routine applications are ensured by the availability of portable 99 Mo/ 99m Tc generators in which 99 Mo is bound as molybdate anion to alumina columns.Current global interruptions of 99 Mo supply that involved uranium fission of highly enriched 235 U targets, aging reactors, and the staggering costs of their maintenance, focused on the search for alternative method of the 99m Tc production [5].One of the possibilities is to replace the reactors with particle accelerators, aiming at a regional production and distribution.At Legnaro laboratories of the Italian National Institute for Nuclear Physics (INFN), a feasibility study related to accelerated-based direct production of 99m Tc by the 100 Mo(p,2n) 99m Tc reaction [6,7] has started since 2011.Theoretical investigations and some recent preliminary irradiation tests on 100 Mo-enriched samples point out that both the 99g Tc/ 99m Tc ratio and 99m Tc specific activity will be basically different in the final acceleratorproduced Tc with respect to generator-produced one, due to the concomitant production of Tc contaminant nuclides, such as 99g Tc, 98 Tc, 97m Tc, and 97g Tc.In particular, the amount of the ground-state long-lived  − emitter 99g Tc, useless for diagnostic procedures, might have a negative effect in the radiopharmaceutical procedures going to compete with 99m Tc for the formation of the corresponding chemically identical radiopharmaceuticals.The presence of an excess of 99g Tc might be responsible for a value of radiochemical purity lower than the standard required for some radiopharmaceutical preparations.In fact, the 99g Tc present in solution could consume reagents of reaction, and in particular the reducing agent (SnCl 2 ).As a result, unreacted [ 99m TcO 4 ] − may remain in the solution, or radioactive by-products not useful for the specific diagnostic procedure may be formed.The quality of 99m Tc is then fundamental for the assurance of radiopharmaceuticals quality [8][9][10].The aim of this work was therefore to perform a set of measurements with 99m Tc, eluted from a standard 99 Mo/ 99m Tc generator, in order to first check possible impact of different 99g Tc/ 99m Tc isomeric ratios on the preparation of different Tc-labeled pharmaceutical kits.Results on both radiochemical purity and stability studies (following the standard quality control procedures) are reported for a set of widely used pharmaceuticals (i.e., 99m Tc-Sestamibi, 99m Tc-ECD, 99m Tc-MAG3, 99m Tc-DTPA, 99m Tc-MDP, 99m Tc-HMDP, 99m Tc-nanocolloids, and 99m Tc-DMSA).These pharmaceuticals have been all reconstituted with either the first [ 99m TcO 4 ] − eluate obtained from the 99 Mo/ 99m Tc generator (coming from two different companies) or eluates after 24, 36, 48, and 72 hours from last elution.

Materials and Methods
The preparation of radiopharmaceuticals reported in All generators, with 99 Mo calibrated activity of 10 GBq, were eluted with 5 mL of saline solution as indicated by each manufacturer.From each generator, we analyzed and compared the three first elutions, performed just after generator delivery (time elapsed between manufacturing and first use can be estimate in 2-3 days), and 3 elutions were carried out after 36, 48, and 72 hours from the previous elution. 99Mo/ 99 Tc Generator Eluates.Generator eluates have been subjected to all the tests [11] required by European Pharmacopoeia [14], and Italian Pharmacopoeia, 12th edn., Norme di Buona Preparazione dei Radiofarmaci per Medicina Nucleare, All.A, p.to A.2 "Generatore di 99 Mo/ 99m Tc (molibdeno/tecnezio)".

Generator Elution Yield.
It was expressed as % of the ratio between the eluate radioactivity measured immediately after elution using a dose calibrator (  ) and the theoretical radioactivity (  ) calculated on the basis of the date of calibration and multiplied by the factor 100 [3].The elution efficiency should be within the range of 90%-110%.

Eluate Visual Inspection.
All eluates were visually inspected, pulling the vial from its shielded container.The operation was performed within an adequately shielded cell for radiopharmaceuticals manipulation; the vial containing the pertechnetate eluate was manipulated by operators using a pair of pliers to guarantee an adequate distance from the hands of the operator.

Eluate pH.
Generator eluate is itself a preparation for injection; ideally it should have a pH as close as possible to the physiological, between 7 and 8.The Pharmacopeia requires that eluates have pH values within the range of 4-8.Since the molybdenum is adsorbed onto the alumina in an acid environment, the pH values of eluates are slightly acid (4.5-6).It was measured by means of pH usual indicator strip (range 0-14) and checked by electronic pH-meter.

Aluminum Content.
It was determined by a semiquantitative procedure employing indicator strips (Tec-Control Biodex Medical, New York, USA) together with standard aluminium solution [12,13].A drop of standard solution with a concentration less than 5 g mL −1 of aurintricarboxylic acid was deposited on indicator paper; subsequently, by side a drop of eluate was deposited.If the coloration produced by the latter is lower than the one produced by the standard solution, it can be assumed that the concentration of aluminium in the eluate is less than the maximum acceptable level of 5 g mL −1 provided by Official Pharmacopoeia [14].

Radionuclidic Purity.
Radionuclidic purity is the percentage of total radioactivity which may be attributed to the daughter radionuclide.In the case of fission-produced generator, the largest potential source of contamination that could exceed the minimum value detectable can be due to the parent ( 99 Mo) [15].Trace amount of other fission impurities [16,17] may be usually present in negligible amounts.The early and quick evaluation of the eluate content of 99 Mo was provided by the following procedure, which involved the use of a lead shield of appropriate thickness (0.6 mm of lead) in order to attenuate 99m Tc emission [18].The activity contained in the unshielded elution vial was measured with a dose calibrator (PET-dose, Comecer, Castelbolognese, Italy); for measuring 99 Mo activity, the elution vial was then placed within the lead shield and its activity was recorded.The thickness of the shield was enough to largely attenuate the 140 keV photons [19], and only partly those greater than 700 keV.The measured activity, multiplied by a suitable correction factor which accounts for the attenuation of 740-780 keV photons due to the lead shielding, provides an estimation of the 99 Mo activity.This value should not exceed 0.1% of the 99m Tc activity according to the European Pharmacopoeia.
For a more accurate determination of the radionuclidic purity [17,20], the same eluate sample was examined again (reassayed) after 7-15 days by means of high-resolution gamma spectrometry using a solid-state, high-purity germanium detector [16].
2.1.6.Radiochemical Purity.It was checked by paper chromatography, Whatman no.1 paper strips and saline as mobile phase.According to this procedure, 99m TcO 4 − migrates with the solvent front (  = 1), whereas reduced hydrolyzed 99m Tc remains at the origin (  = 0).The radioactivity distribution was measured by a scanning radiochromatography detection system for thin layer chromatography (Cyclone instrument equipped with a phosphor imaging screen and an OptiQuant image analysis software (Packard, Meridien, CT)).Eluate radiochemical purity should be greater than 95%.
2.1.7.99 Tc to Active 99 Tc Ratio. 99Mo decays to 99g Tc (12.4%) and 99m Tc (87.6%), and the latter, with a physical  1/2 of 6.0067 h, decays to 99g Tc ( 1/2 = 211,100 years).Due to this particular branching decay of 99 Mo, even fresh elutions from a generator always contain both isotopes ( 99m Tc and 99g Tc), indistinguishable from the chemical point of view.The amount (g) of total technetium present in the eluate is directly related to the amount of 99 Mo atoms present on the column (i.e., 99 Mo activity) and the time that elapsed since the previous elution.The total number of Tc atoms, namely, the sum of 99g Tc and 99m Tc, has been calculated as follows: where  ∘ 99 Mo is the initial 99 Mo atoms number present on the column,  1 is the decay constant of 99 Mo (0.0105 hours −1 ), and  is the time that elapsed since the last elution.The number of 99m Tc atoms has been calculate as follows: where  2 is the decay constant of 99m Tc (0.1149 hours −1 ) and  is the decay probability ( = 0.876).A simplified decay scheme of 99 Mo to 99g Tc is shown in Figure 1.From the above equations the number of 99g Tc atoms can be easily calculated as follows: and thus the 99g Tc to active 99m Tc ratio can be estimated.The determination of the 99g Tc content in a fresh eluate requires an immediate measurement after the elution of the 99m Tc activity and a later measurement of the total activity of 99g Tc (in a few months almost all the 99 Mo and 99m Tc atoms decay into 99g Tc).The evaluation of 99m Tc activity in the sample has been performed by using a dose calibrator (PET-dose, Comecer, Castelbolognese, Italy), while the evaluation of 99g Tc activity has been performed using the TRI-CARB 2810TR liquid scintillation analyzer (Perkin Elmer Inc., Monza, Italy).The samples for 99g Tc activity measurements were prepared taking an aliquot of 0.8 mL from an eluate decayed for 60 days (total volume of the eluate: 5 mL) and adding 5.4 mL of liquid scintillator (Ultima Gold LLT cocktail, Perkin Elmer Inc., Monza, Italy).The measurement of 99g Tc activity was performed using the 0-295 keV energy window.

Radiopharmaceuticals
Labeling.The elutions were used to label different commercial kits (Table 1).Kits reconstitution was performed according to the methods described in the package included within the commercial kits.The radiochemical purity (RCP) of radiopharmaceuticals was evaluated immediately after preparation ( = 0) and at the end of the stability period indicated by the manufacturer.The radiochemical purity and stability were measured using methods specified by manufacturer, with the exception of TechneScan (Mallinckrodt) for which the following chromatographic system was used [21]: mobile phase, 54/45/1 (physiological/methanol/glacial acetic acid) and stationary phase, RP-18 (Merck).Thin-layer chromatography plates were analyzed with a Cyclone instrument equipped with a  Tc-pertechnetate solution, and the data have been acquired with the YAP-(S)PET small animal scanner prototype [22] and reconstructed by using an EM-ML algorithm.

Results and Discussion
The results of the quality control (Table 2) performed on all eluates obtained from two different generator (DRY-TEC GE Healthcare and Elumatic III IBA) are consistent with the European Pharmacopoeia requirements [12].For simplicity, data of visual inspection, yield of elution, and the aluminum content in eluates are not reported, because they fell within European Pharmacopoeia requirements.
The radiochemical purity (RCP) values of all radiopharmaceuticals labeled with each eluate are reported in Tables 3 and 4. Results refer to the RCP evaluated immediately after the preparation ( = 0).For simplicity, data at the end of the stability period specified by the manufacturer are not reported, because they fell within the specifications required by the manufacturer.Tables 5 and 6 report the RCP data obtained from reconstitution of the kits with the first eluate.The values refer to the checks carried out immediately after the preparation ( = 0) and at the end of the stability period specified by the manufacturer in the package insert of each radiopharmaceutical.The values of radiochemical purity are always superior to the standards required by the manufacturer.The results show that the total amount of technetium ( 99g Tc + 99m Tc) present in the first eluate and in the eluates obtained at longer intervals, from 24 h up to 72 h, did not affect the radiochemical purity of the final products.Table 7 shows an estimation of the total amount of technetium present in an eluate obtained from a 99m Tc generator with 99 Mo calibrated activity of 10 GBq.The ratios  of three 99m Tc eluates at 24 hours and two 99m Tc first eluates at 48 hours have been measured, and the results have been  24 h = 3.23 ± 0.15 and  48 h = 6.68 ± 0.31, respectively.While     the experimental value of first eluates at 48 hours is in good agreement with thetheoretical value of 6.5, the experimental value of eluates at 24 hours shows a large difference with respect to the theoretical value of 2.55.This discrepancy could be explained by taking into account the elution efficiency  = 0.91 of 99 Mo generators used in our work.Indeed, the recalculated ratio  at 24 hours is included in the range (2.78-3.38)and depends on temporal sequence of previous elutions.The reconstructed SPECT images of NEMA phantom, for the different 99g Tc/ 99m Tc ratios  used, are shown in Figure 2. The average reconstructed activity along the phantom axis, for the three values of , is shown in Figure 3.The visual inspection on the images doesn't show significant difference in image quality and radioactivity distribution.Currently, CERETEC is the only commercial product for which the use of a fresh eluate, obtained from a generator eluted for not more than 24 hours, is required.This exception is linked to the low amount of tin chloride dehydrate in its formulation (7.6 g), which makes its radiochemical purity strongly influenced by the amount of 99g Tc present in the eluate.All formulations studied possess significantly higher amount of tin.A further limitation to the use of eluates characterized by greater amount of 99g Tc (elution intervals > 24 h) could be due to radioprotection reasons related to the physical characteristics of 99g Tc ( 1/2 = 2 × 10 5 y,  max = 292 keV).However the amount of radioactivity associated with the mass value is very low (e.g., 1 g of 99g Tc corresponds to 630 Bq).

Conclusion
In order to first check the possible impact of different 99g Tc/ 99m Tc isomeric ratios on the preparation of different Tc-labeled pharmaceutical kits, the radiochemical purity and stability of 99m Tc-Sestamibi, 99m Tc-ECD, 99m Tc-MAG3, 99m Tc-DTPA, 99m Tc-MDP, 99m Tc-HMDP, 99m Tcnanocolloids, and 99m Tc-DMSA were studied by using 99m Tc eluates coming from 99 Mo/ 99m Tc generator eluted at different times from the previous elution.The results prove that radiochemical purity and stability of these radiopharmaceuticals are not affected up to 99g Tc/ 99m Tc ratio of 11.84.
A future goal will be to repeat the experiments with 99m Tc eluates coming from generators with 99 Mo calibrated activity higher than 10 GBq, in order to check the possible impact of 99g Tc in higher 99m Tc activities solutions at different 99g Tc/ 99m Tc ratio.
Another future goal will be to study the impact of accelerated-based 99g Tc and other Tc-isotopes on the image quality and determine the allowed limit for 99g Tc and other Tc-isotopes in the final accelerator-produced Tc.

Table 1 :
Radiopharmaceuticals used in the study.

Table 2 :
pH,99Mo,103Ru, and 131 I amounts and radiochemical purities (mean ± standard deviation) of the evaluated generators' elutions.European pharmacopoeia; 36, 48, and 72 h indicate the time from the previous elution; the amount of 99 Mo represents the % of total radioactivity in the generators' eluates determined with "rapid method"; 103 Ru (0.497 MeV) and 131 I (0.365 MeV) are the most common gamma radionuclide detectable by gamma spectrometry, and in the table are reported the % of total radioactivity in the generators' eluates.

Table 3 :
RCP of radiopharmaceuticals at  = 0, prepared with generator DRYTEC GE Healthcare eluates at time superior to 24 h from the last elution.Three 99m Tc eluates produced by 99 Mo/ 99m Tc generator with different 99g Tc/ 99m Tc ratio  were used for imaging studies; the  values were 4.16, 9.51, and 15.2, respectively.Each tomographic acquisition has been performed by filling a NEMA phantom NU 4-2008 with 74 MBq of 99m

Table 4 :
RCP of radiopharmaceuticals at  = 0, prepared with generator Elumatic III (IBA) eluates at time superior to 24 h from the last elution.RCP 36,48,72 indicate the radiochemical purity carried out with eluates obtained 36, 48 and 72 hours after the previous elution.

Table 5 :
RCP of radiopharmaceuticals at  = 0 and at the expired time specified by the manufacturer, prepared with the first eluate obtained from DRYTEC GE Healthcare generator.RCPex indicates the radiochemical purity at the end of the expired time.

Table 6 :
RCP of radiopharmaceuticals at  = 0 and at the expired time specified by the manufacturer, prepared with the first eluate obtained from Elumatic III (IBA) generator.

Table 7 :
Evaluation of total technetium amount in 99m Tc eluates coming from a generator with 99 Mo calibrated activity of 10 GBq, at different times by previous elution.