Comparative study of different chromatographic methods for quality control of113mIn-radiopharmaceuticals

Paper chromatography and Gel Chromatography Column Scanning technique (GCS) have been applied for the separation of indium fractions in^113mIn-radiopharmaceuticals. By these techniques the percentage of ionic indium,^113mIn-colloid and^113mIn-compound have been determined. The resolution efficiency of the gels was found to be significantly influenced by the gel type media and the pH of the eluent. The results obtained from the GCS-profiles indicated that the Sephadex G-50 Fine was the best and can be routinely used in the radiochemical quality control of the^113mIn-phytate. Good separation of^113mIn-colloids,^113mIn-microaggregate and^113mIn-phytate from carrier-free^113mIn-eluate was performed using Whatman No. 3, previously washed with 0.04N HCl and developed either with 0.9% NaCl (for^113mIn-colloid), or 85% methanol (for^113mIn-phytate), or phosphate-methanol buffer (for^113mIn-microaggregate) as rapid and simple procedure for determination of the radiochemical quality control of indium compound in the forms of radiocolloids.     

Comparative quality control of some bone-seeking99mTc radiopharmaceuticals by gel chromatography

The radiochemical purity of the three osteopatic ligands:^99mTc(Sn)-PyP,^99mTc(Sn)-DPD and^99mTc(Sn)-MDP has been determined by gel chromatography on Sephadex. The results of the analyses strongly depend on the composition of the eluent. The dilution effect of pure saline as eluent was observed in all the preparations examined. The most sensitive was found to be^99mTc(Sn)-PyP. The retention of^99mTc activity bound to the gel matrix (^99mTc-hydrolyte) was over 30%. The diphosphonates were found to be more stable (retention 10–15%). The retention is substantially lower, i.e. a high recovery of the labeled complexes is obtained when the eluent contains the ligand. The best results are obtained when the eluent contains the same concentrations of ligand and reductant as in the labeled complex. There was no significant difference in the behavior of the given radiopharmaceuticals prepared as a fresh solution and in the freeze-dried kit.     

Radiochemical quality control of99mTc-phytate

Several chromatographic methods have been used for determining the radiochemical purity of^99mTc-phytate. Good separation of^99mTc-phytate from radiochemical impurities was performed using gel column chromatography packed with Sephadex G-10. Reduced^99mTc-complexes and^99mTc-phytate were not separated by paper and thin layer chromatography.This work has been presented at the Fifth International Symposium on Radiopharmaceutical Chemistry, Tokyo, July 9–12, 1984.     

Statistical analysis of the results of99mTc-MDP quality control

The methods used for control of radiochemical purity of^99mTc-MDP are presented. TLC method on silica gel, developed with methanol and acetone (11 v/v), was convenient for determination of^99mTcO ₄ ^– with the content of 2.6±1.2%. The reliable results on detection of^99mTc hydrolyzate (2.2±1.3%) and for another^99mTc-MDP complex (13.2±2.8%) were obtained by application of ITLC (SA), developed with Sn-MDP. By Sephadex G-25 column chromatography (1.5 cm×5 cm) the separation of^99mTcO ₄ ^– was not achieved. The range of normal^99mTc-MDP biodistribution values in the organs of experimental animals have been determined. The mean value of bone distribution was 8.4±1.13%/g, in muscles 0.071±0.033%/g, while uptake in liver and kidneys was below 5%. Chi-square test and P show that the results on biodistribution of^99mTc-MDP in liver, bones and muscles are arranged around their mean values, which is statistically allowed.     

Characteristics and properties of some Albanian99mTc-kits

The Radiopharmaceutical Group have undertaken a program to study and develop methods for production and quality control of different kinds of^99mTc-kits. Here we present some results and properties for pyrophosphate, gluconate, phytate, DTPA, HEDPA and MDP kits. Work is under way to develop others' radiopharmaceuticals and^99mTc-kits.     

Characteristics and properties of some Albanian99mTc-kits

The Radiopharmaceutical Group have undertaken a program to study and develop methods for production and quality control of different kinds of^99mTc-kits. Here we present some results and properties for pyrophosphate, gluconate, phytate, DTPA, HEDPA and MDP kits. Work is under way to develop others' radiopharmaceuticals and^99mTc-kits.     

Application of macroautoradiography and instantimage in radiopharmaceutical research

The evaluation of the pharmacokinetics of pure, beta radiopharmaceuticals is not possible with scintigraphy. Both whole-body autoradiography (WBAR) and Packard Instantimager, a device for rapid imaging, were used to study the whole body distribution of γ- and β-emitting radionuclides (^99mTc-MDP,^99mTc-sulfur colloid, or¹⁸⁸Re-HEDP), and results obtained from both methods were compared. The biodistribution of^99mTc-MDP and¹⁸⁸Re-HEDP were seen mainly in bones including skull, spine, and vertebrae of spine and cardiac and skeletal muscles. The^99mTc-sulfur colloid localized in liver, bone marrow, and spleen. The resolution of WBAR is superior than that of Packard Instantimager. However, the advantage of Packard Instantimager is that rapid imaging within few minutes is possible with it, while WBAR imaging requires several hours to days.     

Preparation of99mTc-bleomycin with electrogenerated tin(II) ions

Some parameters influencing the efficiency of labelling of bleomycin with^99mTc using tin electrodes for electrolytic production of tin(II) ions were investigated. Different analytical techniques for quality control of^99mTc-bleomycin were applied. From the results obtained, it has been concluded that gel filtration on Sephadex G-25 can give applicable information about the quality of this radiopharmaceutical.     

Quality control and statistical analysis of biodistribution of commercial99mTc-IDA derivatives

The results obtained for radiochemical purity of ITLC (SA) and (SG) using different solvent systems and low voltage electrophoresis are presented in the paper. Radiochemical purities obtained for^99mTc-dimethyl IDA (^99mTc-HIDA) and^99mTc-diethyl IDA (^99mTc-EHIDA) are 98.1±0.6% and 98.7±0.5%, respectively. Variable^99mTc hydrolyzate contents, depending on the ionic strength of the eluents and on the time interval between labelling and analysis, have been obtained by Sephadex chromatography. The eluent containing Sn-EHIDA inhibits dissociation of^99mTc-EHIDA due to dilution of the preparation during elution of the column and yielding only a small percent of Sephadex bound fraction, as compared to other investigated eluents. The range of normal^99mTc-IDA biodistribution values in the organs of experimental animals and statistical significance of the difference between these two preparations have also been determined. The results obtained for^99mTc-HIDA and^99mTc-EHIDA in the liver are 33.9±5% and 25.7±4.7%, respectively p<0.01.     

Oxidation states of technetium in diphosphonate complexes

Valence states of technetium in two most prominent bone imaging agents. i.e., their model complexes with⁹⁹Tc (⁹⁹Tc-MDP and⁹⁹Tc-DPD) were determined by redox potentiometric titration. Titrations of⁹⁹Tc-pertechnetate by Sn(II) solutions were performed in the presence of a ligand (MDP or DPD), as well as without them. Tc(III) was obtained in complexes with MDP and DPD in strongly alkaline medium (pH 12–13) while in the absence of the ligands Tc(IV) was formed. In strongly acidic medium (pH 2–3) the redox process resulted Tc(IV) in both complexes, while Tc(III) was formed in the absence of the ligands. At pH values near neutral or exactly neutral (important for radiopharmaceutical complexes^99mTc-MDP and^99mTc-DPD), it was possible to perform titrations in the ligand absence only at pH values from 7 to 11.8 (due to in hydrolysis) while the resulting oxidation state of Tc in the presence of ligand depended on the used ligand: Tc(IV)-MDP (pH 5.4–5.9 and pH 7.0–7.4), and Tc(III)-DPD (pH 8.0).     

Radioanalytical studies of99mTc-labelled colloids and macromolecules with gel chromatography column scanning technique

The gel chromatography column scanning (GCS) method was used for the present radioanalytical study of^99mTc labelled colloids and macromolecules. The sample to be analyzed was applied at the top of a column filled with gel to a height of 300 mm. Gels investigated included Sephadex G 25, Sepharose 2B, 4B, 6B (Pharmacia Fine Chemicals, Uppsala, Sweden) and BioGel A-150m (Bio Rad, Richmond, Cal., USA). Initially the gels were analyzed according to their chromatographic column caracteristics both for^99mTc pertechnetate and for colloids. A standardized technique was then used to compare colloids labelled in different ways with^99mTc. The column was developed with 10.0 ml 0.9% NaCl solution. Thereafter the column, which still retains all the radioactivity, was scanned with a slit collimated NaI(Tl)-crystal detector. The scanning profile gives information on the size distribution of the labelled colloids and the presence of other^99mTc-labelled species in the preparation. The above method has been used for comparative studies of various^99mTc-sulfur colloids and^99mTc−Sb-sulfur colloid (Philips-Duphar). Colloid formation has also been studied by electrolytically labelling human serum albumin.     

Biodistribution of the bone imaging agents99mTc-DHPE and99mTc-MDP in rats

The self life of the freeze-dried formulation kits utilized for the preparation of the new bone imaging radiopharmaceutical^99mTc-1,2-dihydroxy-1,2-bis/dihydroxyphosphinyl/ethane /^99mTc-DHPE/ has been investigated as well as the toxicity of the DHPE. In a biodistribution investigation of^99mTc-DHPE in rats, in comparison to^99mTc-methylenediphosphanate /^99mTc-MDP/,^99mTc-DHPE exhibited a certain extent higher skeleton uptake, a higher blood clearance rate, a very low concentration in other organs, a satisfactory biological stability and excretion primarily through the kidneys. These properties demonstrate that^99mTc-DHPE is a new promising skeleton imaging radio-pharmaceutical.     

The stability of99mTc phosphorus compounds in plasma both in vivo and in vitro

The in vivo and in vitro stability of^99mTc hydroxyethlylidene diphosphonate, 99^mTc methylenediphosphonate and^99mTc pyrophosphate in plasma has been studied using paper chromatographic technique as the analytical tool. The results indicate that the amounts of^99mTc activity found both at the origin and R_f range of^99mTcO₄ ^? for in vivo experiments are slightly greater than those for either in vitro or control experiments. However, this amount of^99mTc activity represents about 0.16–0.4% of the injected dose. Therefore, it is suggested that^99mTc phosphorus radiopharmaceuticals are stable in vivo and neither oxidation nor hydrolysis of these bone imaging agents occurs in the blood.     

Quality control and clinical application of two bone imaging99mTc radiopharmaceuticals

The radiochemical purity of MDP and HEDP has been determined by means of gel chromatography on Sephadex and thin layer chromatography on plastic foil silica gel. The comparison of the two radiopharmaceuticals shows equal level of complex formation with^99mTc. The biodistribution demonstrated that the application of HEDP allows earlier scanning than MDP. MDP and HEDP show equal effectivity during the clinical investigations. There is no significant difference in the radiochemical purity within six hours after the reconstitution of the freeze-dried kits. HEDP kit demonstrates shorter period of accumulation and equivalent complex formation levels, so it can be used in routine nuclear medicine diagnostics together with MDP kit.     

Studies of the chemical and biological properties of the bone and acute myocardial imaging agent99mTc-PYP

Correlation between the in vivo distribution and the chemical formulation of^99mTc-PYP complex has been studied. We chose mice to evaluate in vivo biodistribution and gel chromatography column scanning technique for radiochemical analysis. The influence of the pH, Sn(II), pyrophosphate concentration and molar ratios of Sn: PYP on the labelling of pyp with^99mTc has been investigated in vitro and in vivo. The reaction between^99mTc and Sn-PYP was complete within a few minutes. The stability studies were evaluated against dilution. Induced myocardial infarction was evaluated in rats. The clinical evaluation showed excellent definition of sternum and ribs with little blood background activity with normal subjects. Discrete localization of abnormally high activity was shown in the site of recent infarction of the left ventricular myocardium.     

Effect of cuprous ion on labeling of three skeletal imaging agents with99mTc

The effect of increased content of copper on the radiochemical composition of three skeletal imaging agents:^99mTc(Sn)-methylene diphosphonate (MDP),^99mTc(Sn)-pyrophosphate (PYP) and^99mTc(Sn)-2,3-dicarboxypropane-1, 1-diphosphonate (DPD) was observed only in the case of^99mTc(Sn)-MDP. It was found that the radiochemical purity of this radiopharmaceutical falls to about 50% when the copper content reaches about 10^–5 mol dm^–3. According to the results of radiochemical and biological analyses, it could be concluded that with the increase of copper content, the content of free pertechnetate rises, too. The two other radiopharmaceuticals,^99mTc(Sn)-PYP and^99mTc(Sn)-DPD, were found to be stable under the given experimental conditions.     

Kinetic analysis of99mTc-d, 1-HMPAO decomposition and the method of stabilization

The kinetics of^99mTc-d, 1-HMPAO decomposition is studied using home kits. The results showed that^99mTc-d, 1-HMPAO decomposition is a first-order reaction. The decomposition constant k is found to be 0.017±0.007h^–1 under the experimental conditions of 20°C, 185MBq/ml, pH 7.0. The stability of^99mTc-d, 1-HMPAO is affected not only by pH and radioactive concentration, but also by temperature. Using Immol/l gentisic acid as a stabilizer, 740MBq/ml of^99mTc-d, 1-HMPAO can be stabilized for 3h with the radiochemical purity above 80%.     

Effect of the eluent composition in gel chromatography of99mTc (Sn)-methylene diphosphonate

Gel chromatography of^99mTc(Sn)-MDP on Sephadex has been performed by using the eluents of different composition and pH. Samples with constant ligand/reductant ratio were eluted by pure physiological saline as well as by saline which contained either the ligand only or both the ligand and the reductant. Their concentrations in the eluent were the same as those used in the preparation of the labeled complex.     

The interactions of99mTc phosphorus radiopharmaceuticals and human serum proteins

Dialysis and precipitation methods have been used to study the binding affinity of selected technetium-99m phosphorus radiopharmaceuticals to human serum proteins. The binding affinities of three different^99mTc bone imaging agents were found to be inversely related to their respective clearance rates from blood in vivo. The binding order showed^99mTcPPi>^99mTcHEDP>^99mTcMDP. The^99mTc phosphorus radiopharmaceuticals were bound primarily to alpha globulins. The results suggest that the binding of^99mTc phosphorus radiopharmaceuticals to human serum proteins in blood is largely determined by their affinities to the alpha globulins.     

99mTc-dimercaptosuccinic acid: Analytical procedures for its radiochemical quality control

A procedure for the radiochemical purity control of^99mTc-(2,3-dimercaptosuccinic acid) (DMSA), used as a renal scintigraphic agent is described. The proposed chromatographic system entails the use of two successive solvents, first MEK and second aqueous solution of 5% glycine, on the same supporting medium Gelman ITLC-SG. The procedure is fast and leads to separation and estimation of free pertechnetate, hydrolyzed form of^99mTc and^99mTc — DMSA. This system is superior to the others reported in the literature as the spots of the different species are more distinguished and more concentrated. Its reliability has been studied using dimercaptosuccinic acid kits of different manufacturers and the results have been checked biologically.