Analytical and radioanalytical quality control in high specific activity radiotracer preparation

Jointly published by Elsevier Science S. A., Lausanne and Akad#miai Kiad6, Budapest

Journal of Radioanalytical and Nuclear Chemistry, Articles, Vol. 193, No. 1 (1995)39--47

ANALYTICAL AND RADIOANALYTICAL QUALITY CONTROL HIGH SPECIFIC ACTIVITY RADIOTRACER PREPARATION

IN

M.GALLORINI*,M.BONARDI**,C.BIRAT'I'ARI**,F.GROPPI**,S.SAPONARO** *Centrefor Radiochemistry and Activation Analysis, CRAA, CNR and INFN Pavia, via Taramelli 12, 27100 Pavia, Italy **Accelerators and Applied Superconductivity Laboratory, LASA, University and INFN Milano, via F.lli Cervi 201, 20090 Segrate (Milano), Italy

High specific activity radiotracers, used in studies related to trace dements and human health, must be characterized by the following specific requirements: (i) high specificactivity (activity/massof isotopic carrier), (ii) high activity concentration (activity/volume or mass of substrate), (iii) radionuclidic, radiochemical and chemical purifies, (iv) biological compatibility (physiological pI-I, sterility and physiological values). For this purpose, selective radiochemical separations and quality control procedures have been developed and tested at our laboratories for the production of several NCA radiotracers.

The possibility of using high specific activity radiotracers to carry out in-vivo and in-vitro experiments represents a very powerful tool in all those studies aimed at assessing the impact on human health from elements and compounds of environmental and toxicological importance. These radioanalytical procedures allow us to follow and to understand processes such as accumulation, distribution, metabolic patterns and biotrasformation of chemical species. In other words, many of the interactions arising when toxic or pollutant species impact with living organisms can be thoroughly investigated if the corresponding radiolabelled compounds are available in the correct chemical form. 1,2 This, of course, is a very difficult task since the preparation of these "radio-species" requires a wide range of specific expertise (nuclear, chemical, physico-chemical, organo-chemical, biological, biochemical, medical and others), that can be available only in an interdisciplinary team. However, referring to a more restricted problem, such as the case of a long term-low level exposure (TE-LLE) to trace metals resulting from environmental pollution, a series of radioelements were obtained in our laboratories to perform these studies. In this case, high specific activity radiotracers have to be prepared in NCA (No Carrier Added) form, since the real basal concentration in the living organisms is of the order of a few nanograms per gram (ppb). Once the Nearly Carder Free conditions (NCF) are found and tested, using the most appropriate radiochemical separations to isolate the radiotracer from the irradiated target, a series of quality assurance procedures must be applied. In particular, the oxidation state and the chemical form must reflect those of the species under investigation. The biological compatibility, such as pH and sterility, and both chemical and radiochemical purities have to be assured in order to perform the experiments with no modifications of the natural conditions. What follows presented in some detail is a review of the quality assurance procedures and control tests developed and used to check the radiochemical separation yield from the irradiated targets, the radionuclidic, radiochemical and chemical purities, the stability of the radiolabelled chemical species and their specific activity. In addition, examples of the applications of these controls to some radiolabelled species are also reported.

0236-5731/95/US $ 9.50 Copyright 9 1995 Aka~gmiai Kiadr, Budapest All rights reserved

M. GALLORINIet al.: ANALYTICALAND RADIOANALYTICAL

Experimental The irradiations were carded out at the following facilities: the AVF Cyclotron of the University of Milano, the Scanditronix MC40 Cyclotron of JRC-Ispra of the EU and the TRIGA MARK II Nuclear Reactor of the University of Pavia (Italy). Very high purity target materials were used for the radioisotope preparations (Goodfellow Metals and J&M-UK). The radiochemical separations were performed using electronic grade chemicals from Carlo Erba (Italy) and BDH Italia (Italy). Chelex-100 resin, purchased from BIO-RAD (USA), Sephadex G25 from Pharmacia (Sweden), Whatman n.1 Paper from Whatman (USA) and TLC plates from Merck (FRG) were used for gel filtration and radiochromatographic tests. For liquid scintillation counting, liquid scintillation cocktails Ready Gel from Beckman (USA) and Hionic Fluor, Pico Acqua and Ultima Gold from Packard (USA) were used. High resolution gammaspectrometry was carded out with Ge(Li) and HPGe detectors (ORTEC, USA) coupled to computerized MCAs (EG&G-ORTEC, USA). Neutron irradiations for activation analyses were made at the TRIGA MARK-II reactor of Pavia, with a neutron flux of 1013 rdcm 2 s. Atomic absorption measurements were done with a Varian SpectrAA-30 spectrophotometer. ICP-OES measurements were carried out with a Perkin Elmer 5500 spectrophotometer. Anodic stripping voltammetry and differential pulse polarography were done with a Metrohm Polarecord E505/E608. For liquid scintillation counting (LSC), a Beckman LS5000 TD computerized counter was used with the capability to show the shape of the electron conversion and beta spectra. Metallobiochemical experiments on both laboratory animals and ceil cultures were carried out at the radiochemistry laboratory of the Environment Institute (JRC-Ispra of the EU, Italy).

Results and discussion Radiochemical seoarations control: The radiochemical separations developed were usually based on two main steps, consisting of a preliminary separation of the radiotracer activity from the bulk of the target and a second chemical procedure for the final purification of the radiotracer. After the radiochemical procedure was applied, a series of controls had to be carried out to check its selectivity and separation yield. In Tables 1 and 2, a selected list of the radiochemical separations and the relative control procedures is reported. In the cases illustrated as examples, these were done analyzing the resulting solutions by gamma-spectrometry and elemental analysis techniques, such as GF-AAS, ICP-OES, NAA, ASV and colorimetry. Manganese: The 52gMn was produced by (p,xn) reactions on natural chromium, as reported in a previous work. 3 The radiochemical separation developed and adopted consisted of the following five steps: 1. target dissolution - the Cr target (100 mg) was dissolved by 5 ml of 6N HC1, dried and redissolved in a few ml of 1N HC1 containing 1000 ppm of Fe(III); 2. coprecipitation - the Fe(OH) 3 was precipitated by the addition of an excess of 2.5N NaOH, previously saturated with bromine. The 52gMn was almost quantitatively coprecipitated with Fe(III), while the supernatant with the bulk of chromium(VI) was discharged; 3. extraction - the Fe(III) was extracted by diethylether, while the 52gMn was quantitatively recovered in 6N HC1;

40

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4. oxidation - the solution was oxidized by HClO4 at about 220~ and dried. In these conditions, the residual Cr and the 52gMn were present in the (VI) and (IV) oxidation states respectively; 5. purification - the residue was then dissolved in distilled water at pH 6 and passed through a Chelex-100 column, where the 52gMn was almost complefly adsorbed. The Cr(VI) impurities were quantitatively eluted. The 52gMn was finally recovered by elution with 6N HCI as MnC12. The radiochemical separation yield was greater than 80%, as determined by gammaspectrometry. Gallium: The production of gallium radionuclides (67 and 66) by (p,xn) reactions on natural zinc has been described elsewhere. 4 The radiochemical separation consisted of an extraction of Ga(gI) in isoprophyl ether, after the zinc target dissolution in 7N HC1. After the extraction of Ga(III), the extract was washed with 7N HC1 to eliminate the residual zinc impurities. The resulting 66,67Ga was back-extracted in water, dried and used as Ga cation(Ill) in metallobiochemical studies and, as a labelled citrate complex, for biomedical radiodiagnostics.5 As in the previous example, the overall radiochemical separation yield (>95%) was determined by gamma-spectrometry, while the decontamination factor from Zn was determined by ASV. The results are reported in Table 3. Radionuclidic purity control: In all cases, high resolution gamma-spectrometry was utilised to verify the radionuclidic purity while, in some selected cases, liquid scintillation was used to determine the content of electron conversion and pure beta emitters. In the case of 52gMn, some side reactions on 52Cr and 54Cr were observed leading to the production of the two radioisotopes 51Cr and 54Mn. The first one was used as chromium tracer in the radiochemical separation of 52gMn from the target and, at the end of the separation, its content was less then the detection limit of the I-IPGe detector used for gammaspectrometry. The second one, on the contrary, was still detectable at the end of separation, but it was only 0.3% of thetotal manganese activity. For the 67Ga radiotracer, no other radionuclides (i.e. 65Zn and 66Ga) were detected by gamma-spectrometry in the [67Ga]-citrate, after the radioehemical separation adopted and a proper cooling time for the decay of 66Ga. On the contrary, in the case of toxicological experiments to investigate the trace element contamination occurring during treatment of dialytical patients, a mixture of 66Ga and 67Ga was used to label the AI(III), that might be responsible for possible long term diseases. 6 It is well known that long lived 125I is a possible contaminant for the 123I used for diagnostic purposes. Due to the very low gamma-emission energy of 125I (35 keV), it is advisable to determine this radionuclidic impurity using a spectroscopic technique able to identify electron emitters, such as LSC. In fact, this technique was found very effective to check the content of 1251 in 1231, which is present in the range of 0.1 to 0.01% of the total

activity. Radiochemical ouritiv cowl stability: In all the experiments carded out using selected radiolabelled chemical forms of the trace elements investigated, as well as the radiotracers applied in radiodiagnostics, it was necessary to check their chemical forms. For this purpose, radioanalyfical techniques such as Paper, TLC, Ion- and Gel-f'dtration liquid radiochromatography were used. Furthermore, tests to control the variation with storing time (stability) of the oxidation states and the chemical forms were carded out. As a typical example, in the case of toxicological experiments on cell cultures to investigate the manganese behaviour, radiolabeUed MnC12 was used. 52gMn was routinely tested as 52gMn(II) cation by passing its solution in 6N HCI through a Chelex- 100 column, as previously described. In these conditions, Mn(II) is the only oxidation state that is stable in the absence of strong oxidants. For the radiochemical purity and stability of [67Ga]-citrate, a paper radiochromatography test was performed at different time intervals, to determine the ratio between the citrate form 'and the inorganic one, as a function of pH. A Thin Layer Scanner II CB (Berthold) (scanning 43

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speed ranges from 12 mm/h to 3,600 mm/h) was used, choosing a scanning speed of about 600 mm/h, coupled with 2~ G.M. detector, that was found suitable because of its high resolution. The eluent was Pyridine:Ethanol:Water(1:2:4); at pH 5 and 7, the corresponding Rf were 0.83 +_2.0% and 0.72~.3.4%, with a radiochemical purity of 99.99% and 98% at the starting time and 99% and 97% at the expiration time. In all cases, the radiochemical purity found was greater then that required from the U.S. Pharmacope&7 For 58Co and 57COB12 experiments, the radiotracers were commercially obtained from the Amersham Radiochemical Centre (UK), with the following characteristics: 58Co, as chloride in 0.1M HC1, and 57Co cyanocobalamine (vitamin B12), in acqueous solution containing 0.9% benzyl alcohol. These radioactive species have been used to carry out a speciation study of cobalt present in urine of hard metal workers. 8 The 57COB12 was further purified by chromatography separation on an ion exchange column to separate any possible inorganic form of STCo as impurity. The following procedure was adopted: the initial Chelex-100 in Na+ form (100-200 mesh) was loaded in plastic columns (10 cm height, 0.8 cm diameter) and converted to NI-I4+form by sequentially passing 10 ml of 2N HNO3, 20 ml of 1N NH4OH (at about 60~ and distilled water to a final pH of 6.6; the columns were then conditioned with 0.01N NH4COOCH3 buffer at pH 6.6, that was also used as eluting agent. In these conditions, the organo-cobalt is completely eluted (3 volumes washing) while the inorganic Co forms are retained on the resin. The same procedure was used to perform chemical speciation tests between org./inorg. radioactive Co forms on laboratory animals as well as in-vitro human urine.9 The Chelex procedure was also used to follow the stability with the storing lime of the 57COB12 and the transformation of the inorganic 58(20 during the in-vitro incubation of human urine. This last experiment showed that after 24 hours of incubation time, at 20~ and at 5~ the inorganic 58Co is transformed into organic species with respective rates of 60% and less then 30%. To understand if the Co-organic species produced could be associated with that of the cyanocobalamine, a gel-filtration chromatography, using Sephadex G 25, was performed: aliquots of human whole urine were incubated with 57COB12 or inorganic 58120 and submitted to chromatography as well as the eluted fraction from the Chelex column, obtained after passing the inorg. 58Co incubated urine. In all cases, the 57COB12 and 58Co radioactivities were eluted in a single main peak in the same region of 57Co. Stability tests were also applied to investigate the behaviour of other radiotracers, used in different in-vivo and in-vitro experiments. In Table 4, the stability with time of different chemical forms of 51Cr, 74As and 201TI, as determined by paper, TLC and column radiochromatography, is reported. Chemical ouritv: The chemical purity was checked by analyzing both the possible traces derived from the irradiated targets and the impurities contained either in the target or in the reagents used. Chromium and iron determination in manganese-52g tracer: Since the evaluation of the decontamination factor from the Cr target by gamma-spectrometry of 51Cr presented an insufficient sensitivity, the Cr content in the various fractions was determined by GF-AAS. An additional determination of the Cr content was carded out by INAA. The Fe content, derived from the precipitation steps, was analyzed by GF-AAS. The results are reported in Table 3. ~f Zinc and iron determination in gallium-67 tracer: In the case of medical application of 67Ga, the most relevant impurities to be determined are Zn and Fe.3 In Table 3, the results obtained in the analysis of these elements are reported. In this case, two electroanalytical techniques, differential pulse polarography (DP) and anodic stripping voltammetry (ASV) on a HMDE, were used.

45

M. GALLORINIet al.: ANALYTICALAND RADIOANALYTICAL

Trace elements determinations in vanadium-48 tracer: In the case of vanadium-48 obtained via Ti(p,xn) reactions, used in metabolic studies on laboratory animals10 to assess metallobiokinetics of trace metals derived from fossil fuel combustion processes, the determination of a series of elements and titanium was carried out by INAA, AAS and ICPOES, obtaining the results shown in Table 5. In this specific case it was necessary to verify the absence of trace metals impurities that could interfere in the biochemical equilibria occurring in the 48V uptake and metabolism. Sr~ecffic activity determination: Since the carrier-free conditions are only theoretically reached, the quantitative determination of the corresponding specific activity, analyzing the final radioactive solution, is needed. In Table 3, as examples, the analytical results found for 52gMn, 67Ga and 201TI are reported. The total manganese content was determined by GF-AAS, since its determination by NAP, was prohibited by the interferring neutron reaction on the Fe impurity, that leads to 56Mn production through 56Fe(n,p) reaction. Gallium analysis was performed by ASV while, for the T1 determination, the colorimetric technique with Rhodamine B was found very effective. The specific activities found for many other radiotracers are given in separate works.

Conclusions

The quality control procedures adopted to check the specific characteristics and purities of the radiotracers produced are comprehensive of many different chemical techniques (conventional and radioanalytical). These have to be applied time by time in dependence of each single case. Tables I and II summarise all these procedures developed and used in our laboratories. In all cases, verified quality standards have been obtained and many radiotracers have been prepared in nearly carder-free conditions (NCF). This was very useful for those trace metal toxicological experiments (both in-vitro and in-vivo) where the basal concentrations of investigated metals are of the order of ppb or less.

This workhas beencarriedout with the financialsupportof the 5th Groupof the INFN(PREPURexperimen0 and the MURST:CoordinamentoNazionaledi FisicaBiomedica.

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