Determination of Sn 2 + in Lyophilized Radiopharmaceuticals by Voltammetry , Using Hydrochloric Acid as Electrolyte

O objetivo do estudo foi desenvolver e validar um método de rotina para a determinação específica de Sn em kits de radiofármacos 2-metóxi isobutil isonitrila (MIBI). Para a análise, foi utilizado o equipamento analisador voltamétrico. Experimentos de triagem mostraram que o eletrólito HCl 1 mol L apresentou os melhores resultados entre todas as soluções testadas. Experimentos de estabilidade mostraram declínio gradual na corrente de Sn no MIBI, e 23 dias depois da preparação da solução, a corrente desapareceu. Para confirmar a seletividade de técnica utilizando o HCl 1 mol L, induzimos a oxidação do SnCl2, resultando em um declínio proporcional da corrente no voltamograma. A confiabilidade do método foi observada com os valores de precisão e exatidão intrae inter-ensaios, e com a robustez. Nós proporcionamos novos dados quanto a detecção seletiva de Sn na presença de sua forma oxidada em kits de radiofármacos, utilizando HCl 1 mol L como eletrólito.


Introduction
5][6][7] Other isonitrile-based compounds have been used beforehand, but they presented low stability, having their application discontinued. 8Subsequently, the radiopharmaceutical MIBI proved to be greatly efficacious, overcoming the limitations of previously developed compounds with the same diagnostic purposes. 9he stannous ion (Sn 2+ ), used mainly in the form of SnCl 2 salt, is added to lyophilized radiopharmaceutical kits to allow the complexation to the radioisotope.It is mainly employed in order to promote the reduction of pertechnetate ion (TcO 4 ) -for the preparation of 99m Tc-radiopharmaceutical kits. 10,11For that reason, quality control measurements, involving determination of Sn 2+ , are fundamental in the production of radiopharmaceutical kits.In fact, Sn 2+ might be affected by several factors, such as oxygen and light exposure, generating its oxidized form stannic ion (Sn 4+ ).For instance, dental formulations containing SnF 2 are highly unstable in aqueous solutions, since Sn 2+ can be easily oxidized to Sn 4+ , compromising the physical and chemical properties of the product. 12he implementation of quality control procedures in radiopharmacy is extremely important to ensure that unsuitable products will not be used in patients.It is well known that either lacking or excess of Sn 2+ in lyophilized formulations might affect the radiochemical purity, as well as the quality of images.4][15][16][17] At the present, there are a few techniques available for the selective determination of Sn 2+ , including titrimetric analysis (redox-titration) and voltammetry.The low sensitivity of titrimetric methods for the concentrations of tin used in radiopharmaceutical kits impairs its precise and accurate measurement. 13Thus, especially for radiopharmaceutical kits with low Sn 2+ contents, the only reliable method to properly determine Sn 2+ is voltammetry. 18he peaks obtained in the latter technique give qualitative information through the value of the potential peak E (V), while the quantitative information is provided by the current peak i (A), in the presence of the selected electrolyte. 19,20The determination of Sn 2+ by voltammetry has been performed before using electrolytes containing complex mixtures of salts, buffers, acids and bases, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), hydrochloric acid (HCl), citric acid (C 6 H 8 O 7 ), potassium chloride (KCl) and sodium hydroxide (NaOH). 21,22he aim of this study was to validate a simple method for determination of Sn 2+ in MIBI radiopharmaceutical kits to furnish a reliable routine method for quality control in radiopharmacy.

Apparatus and electrochemical technique
Equipment 757 VA Computrace (Metrohm) was used with a three electrode cell for differential pulse polarography technique: multimode mercury working electrode (MME), platinum auxiliary electrode (Pt) and electrode silver/ silver chloride (Ag/AgCl) reference containing potassium chloride (3 mol L -1 KCl) electrolyte solution.

General procedures
For initial screening of a series of electrolytes, we tested the current and reproducibility of standard solutions at the concentration of 50 mg L -1 .The sample used for analysis was the radiopharmaceutical MIBI containing approximately 0.052 mg of Sn 2+ , corresponding to 17.53 mg L -1 , after suspension of lyophilized kit with 3 mL of distilled and deionized water (dd-water).The standard solution of SnCl 2 used for validation assays was prepared at the same concentration present in the radiopharmaceutical kit (17 mg L -1 ), whilst the standard solution of SnCl 4 pentahydrate, due to the lower sensibility of the assay for Sn 4+ , was prepared in a higher concentration (50 mg L -1 ).The samples and the standards were dissolved in oxygenfree dd-water by nitrogen saturation to avoid oxidation.HEPES (1 mol L -1 HEPES, 1 mol L -1 NaF, 1 mol L -1 NaNO 3 , and H 2 O), 1 mol L -1 KCl, 1 mol L -1 NaOH, 0.1 mol L -1 EDTA, 0.1 mol L -1 KNO 3 , PIPES (1 mol L -1 PIPES, 1 mol L -1 NaF, 1 mol L -1 NaNO 3 , and H 2 O), HCl plus citric acid (0.2 mol L -1 ) and HCl (0.2 mol L -1 and 1 mol L -1 ) electrolytes were tested.In the protocols to induce the oxidation of Sn 2+ , 200 µL of 30% H 2 O 2 were used.
The use of the electrolytes 1 mol L -1 PIPES, 1 mol L -1 NaF, 1 mol L -1 NaNO 3 , and H 2 O were prepared according to the newsletter of Metrohm (CH4-0381-042002), which was composed of 11 mL of deionized water, 7 mL of NaF, 1 mL of 1 mol L -1 NaNO 3 , and 1 mL of 1 mol L -1 PIPES buffer.The electrolyte 1 mol L -1 HEPES was prepared according to the PIPES protocol, replacing PIPES by HEPES.The electrolyte HCl plus citric acid was made by mixing 5 mL of 0.4 mol L -1 HCl and 5 mL of 0.4 mol L -1 citric acid in the vessel, with a final concentration of 0.2 mol L -1 .A volume of 10 mL of the electrolytes PIPES, HEPES, 1 mol L -1 KCl, 0.1 mol L -1 EDTA, 1 mol L -1 NaOH, 0.1 mol L -1 KNO 3 , 0.2 mol L -1 HCl, and 1 mol L -1 HCl were added in the vessel during each analysis.The electrolytes were initially tested with a Sn 2+ concentration of 50 mg L -1 for assessing the current and reproducibility.The analysis was performed in triplicate in order to determine the most suitable conditions concerning deposition time, concentration, and pH of the electrolyte.Those showing stable and reproducible currents were further tested with a Sn 2+ concentration of 17 mg L -1 .
The differential pulse voltammetric analysis was performed under the following conditions: -0.2 V deposition time, 90 s pulse time, 10 s equilibration time, -0.2 V starting potential, -0.55 V end potential, 0.004 voltage step, 0.050 V pulse amplitude, 0.04 s pulse time, and 0.1 s voltage step time.These values are defined by the manufacturer.To assess the possible interference of Cu 2+ , we have used a potential range varying from 0 to -0.55 V. Initially, 10 mL of electrolyte were added to the polarographic vessel, and a flow of 1 kgf cm -2 of nitrogen gas was applied for 5 min.The method of manual standard addition for quantification was adopted.The calibration curve was made by blank determination (electrolyte) and five successive additions of 200 µL (10 mg L -1 of Sn 2+ ) standard solution into the vessel, in order to record the polarographic concentrations of 0, 10, 20, 30, 40 and 50 mg L -1 .For determination of Sn 2+ in the MIBI kits, the samples were resuspended with 3 mL of dd-water and 200 µL of each sample were added into the vessel to obtain the initial voltammogram.Subsequently, two successive additions of 200 µL of standard (17.0 mg L -1 ) were carried out.The analyses were performed in triplicate, and 3 separated samples of lyophilized compound were used.The results were expressed as mean ± standard deviation (SD).

Polarographic method validation
The polarographic method was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, recovery and robustness.

Electrolytes for determination of Sn 2+
The results for HEPES were not satisfactory, as the electrolyte caused interference in the baseline, and showed no peak in the voltammogram at the potential range corresponding to Sn 2+ .For 1 mol L -1 KCl, the same potential was obtained for SnCl 2 and SnCl 4 , according to assessment at 50 mg L -1 , following separate readings, clearly showing that is not possible to separate Sn 2+ from Sn 4+ (Figure 1).The electrolytes 0.1 mol L -1 EDTA and 1 mol L -1 NaOH, using Sn 2+ at 50 mg L -1 , generated erroneous results, due to the decay of the current between the standard additions, as it can be observed in Figures 2a  and 2b, respectively.
For 1 mol L -1 PIPES and a concentration of 17 mg L -1 of Sn 2+ , the obtained result was approximately 13 mg L -1 , giving a recovery of 76%.Similarly, the use of HCl with citric acid provided a reading of approximately 20 mg L -1 , for a standard Sn 2+ solution of 17 mg L -1 (recovery of 118%).A decreased recovery was observed by employing 0.2 mol L -1 HCl as electrolyte, showing a reading of 12 mg L -1 (71%) (Figures 4a-c, respectively).
Considering that 1 mol L -1 HCl electrolyte showed the best reproducibility for determining Sn 2+ when compared to the other solutions, according to assessment of either concentrations of 50 or 17 mg L -1 (Figure 5), this was chosen to proceed validation.Technique validation for determining Sn 2+ for differential pulse polarography using 1 mol L -1 HCl as electrolyte

Stannous ion detection in the presence of stannic ion
The solutions were prepared from vials containing the lyophilized MIBI.The current measurement was performed during three weeks.There decrease of current was observed through this voltammetric technique few days after the preparation of the radiopharmaceutical solution, which was intentionally stored at room temperature and exposed to light (Table 1).Vol. 25, No. 9, 2014   To observe the selectivity of the electrolyte in the solution of SnCl 2 , a reading using freshly prepared SnCl 2 standard was performed.The fast oxidation of Sn 2+ was induced by adding H 2 O 2 , and incubating the solution in water bath at 37 °C for 5 min. 13No current corresponding to Sn 2+ was observed.However, despite the disappearance of the Sn 2+ current, we observed an increase in the baseline current.This fact seems to be due to oxidation of the working electrode mercury drop by the action of H 2 O 2 that could be hiding the Sn 2+ current.To exclude this possibility, another Sn 2+ standard was prepared and incubated for 24 h in water bath for 37 °C, without adding H 2 O 2 .Figures 6a-c, respectively, show the disappearance of the current of Sn 2+ without any change of baseline current following this procedure.
These results show that oxidation of Sn 2+ and the formation of Sn 4+ do not affect the analysis, at the tested conditions.

Sensibility, specificity and selectivity
The limit of detection was determined by adding concentrations of 1 mg L -1 of the Sn 2+ standard.The detection limit of this method was 3 mg L -1 .The limit of quantification was the lowest analyzed amount, which can be measured with defined precision and accuracy and reproducible with a coefficient of variation (CV) up to 20% and accuracy of 80-120%.The limit of quantification values was 4.57 ± 0.49 mg L -1 , with CV of 1.06 % and accuracy of 114.21%.
For determination of specificity, the excipient of MIBI was tested to assess the interference with voltammetric method.The ability of the method to detect Sn 2+ , without interference of excipients in its potential, is depicted in Figure 7.
To assess the interference of copper in the potential of Sn 2+ , we obtained voltammograms with (i) Cu 2+ standard, (ii) Cu 2+ standard with Sn 2+ standard and (iii) radiopharmaceutical MIBI kit under normal conditions, as demonstrated in Figures 8a-c, respectively.
The obtained peaks of copper were detected at a different potential than Sn 2+ (-0.13 V), and all assay tests were free of interference from this element during determination of Sn 2+ .

Precision and accuracy (recovery) intra-and inter-assay
The intra-and inter-day precision and accuracy data are shown in Table 2. Precision was expressed as the percentage of coefficient of variation (CV), and     accuracy was expressed as the percentage of the added concentration.

Robustness
As shown in Table 3, intentional variations were performed in the concentration of electrolyte, deposition time, use of water without nitrogen, and different operators.
The results indicate that the method is robust in relation to the possible variations that may occur during the execution of the technique, including the small variations in the electrolyte concentration and the execution by different operators.However, some specific parameters, such as the deposition time of 90 seconds, and water nitrogenation must be strictly respected.

Linearity
At least three calibration curves were carried out in the range of 10-50 mg L -1 of Sn 2+ (Figure 9).This experimental set presented a correlation coefficient of 0.9993, revealing the linearity of the method.

Discussion
The determination of Sn 2+ concentrations represents a very important step in the quality control procedures Vol. 25, No. 9, 2014 in radiopharmacy, guaranteeing safety and allowing the correct diagnosis. 13In the case of MIBI radiopharmaceutical kits, the exact concentration of Sn 2+ is essential for efficient cardiac perfusion and adequate scintigraphy. 13revious studies published methods for tin determination using technologies such as inductive coupled plasma-mass spectrometry (ICP-MS) 14 and gas chromatography coupled with mass spectrometry (GC/MS). 23,24However, these techniques do not allow selective determination of tin species, considering that Sn 2+ concentrations are calculated on the basis of total tin concentrations.Tin can also be determined by atomic absorption spectroscopy (AAS), but this technique requires complex extraction procedures. 24,25urrently, voltammetry represents a low-cost technique able to precisely detect small concentrations of this ion, both quantitatively and qualitatively.Nevertheless, there are only few studies for this purpose using the MIBI radiopharmaceutical kit, especially when considering the appropriate electrolyte to be used in the selective analysis of Sn 2+ .Thus, we developed and validated a simple voltammetric method for speciation of Sn 2+ in the MIBI radiopharmaceutical, using 1 mol L -1 HCl.
Hubert et al. developed a simple and rapid method for the separation and determination of Sn 2+ and Sn 4+ in tin octoate, a catalyst used in the synthesis of polydimethylsiloxane (PDMS). 24The detection of Sn 2+ at the same potential of Sn 4+ is a problem for nuclear medicine, since Sn 4+ represents an impurity in lyophilized radiopharmaceutical kits.Several electrolytes have been described for the determination of Sn 2+ , but there are differences in the specificities reported.Therefore, many methods used are effective in determining the sum of Sn 2+ and Sn 4+ , but they fail to provide a selective identification of Sn 2+ .Decristoforo et al. described a polarographic method for determination of Sn 2+ in technetium cold kits using a mixture of water, methanol and perchloric acid as electrolyte. 10In addition, Almeida et al. 13 described the selective detection of Sn 2+ (-0.350 to -0.400 V) by using 3 mol L -1 H 2 SO 4 in pyrophosphate (PYRO) and methylene diphosphonate (MDP) radiopharmaceutical kits, although the authors have not investigated the effects of Sn 2+ oxidation in their study.Herein, we tested several electrolytes, demonstrating that a series of complicating factors, such as determination of Sn 4+ at the same potential of Sn 2+ , decrease in current after standard addition, or disproportionate growth of the currents, excluded the use of most options.In our study, among all the electrolytes tested for determining Sn 2+ , the only one showing good reproducibility and specificity was 1 mol L -1 HCl.Moreover, this electrolyte is easily prepared and presented a good stability.The best result obtained among the other electrolytes was achieved with HCl plus citric acid, but this solution presented some problems in the recovery (more than 115%) and linearity.These problems were detected in both determination of standard of Sn 2+ in the excipient and lyophilized radiopharmaceutical kit.This appears to represent a good option for other samples, as described by Pérez-Herranz et al. for tin octoate, although it does not seem to be reliable for MIBI kits. 18o gain further insight on the applicability of 1 mol L -1 HCl, we initially prepared a solution of SnCl 2 for reading, followed by forced oxidation of Sn 2+ with H 2 O 2 .The hydrogen peroxide acts as an oxidizing agent in acid aqueous solution. 26After the reading, we observed a decrease of the current, which was accompanied by an increased  baseline.To rule out the possibility that baseline would hide the Sn 2+ peak, we carried out separate experiments without adding H 2 O 2 , which would be responsible for the increase in baseline, using water bath at 37 °C to oxidize Sn 2+ .Additionally, a decrease of Sn 2+ currents was observed throughout the three weeks of exposition to room temperature and light.Studies show that the temperature is directly related to the oxidation of Sn 2+ . 10,13In this case, Sn 4+ formed even at high concentrations of Sn 2+ , was not enough to interfere with the analysis and recovery of the analyte.According to this analysis, we might affirm that our method using 1 mol L -1 HCl was selective for Sn 2+ quantification.However, when we performed a reading of Sn 4+ using SnCl 4 salt in 1 mol L -1 HCl we observed a peak at the same potential of Sn 2+ .The same does not occur when we performed the readings using SnCl 2 , because the results obtained by us showed that after the oxidation of Sn 2+ to Sn 4+ in solution, the current disappears in the potential where Sn 2+ is detected (-0.40 V), indicating the selectivity of the method for Sn 2+ .We suppose that this occurs because the Sn 2+ oxidized by oxygen in solution (reaction that is favored by the presence of 1 mol L -1 HCl) 27,28 generates complex species of Sn 4+ which cannot be detected at the potential of -0.40 V.The formation of Cl -/Sn complex can display several electrochemical profiles 29 and the formed complex can be responsible for the non-detection of oxidized Sn 2+ in solution.This hypothesis is strengthened by the fact that Sn 4+ complexes display completely different properties in relation to Sn 2+ complexes. 30,31Therefore, these conditions related to the oxidation of Sn 2+ were confirmed by our study because the Sn 2+ oxidized was not detected in the voltammetric analysis.
During the Sn 2+ analysis with 1 mol L -1 HCl, considering all steps to guarantee the quality of SnCl 2 (preparation at the time of use, correct manipulation, ideal storage, and protection against light), it is possible to presume that the read current of Sn 2+ corresponds to the amount weighed for analysis.Altogether, intra-and inter-assay tests on robustness, accuracy and precision, revealed the method presented by us as a simple, quick, and inexpensive voltammetric approach to selectively determine Sn 2+ in lyophilized MIBI kits.However, to maintain satisfactory robustness, accuracy, and precision, it is imperative to maintain the methodological parameters, as well as qualified operators.

Conclusion
Validation results provided by us indicated that the method presented herein shows high specificity for Sn 2+ determination, without interference by Sn 4+ , excipients from formulation of MIBI or copper.The method presented high sensitivity, and might as well be considered a reliable parameter for the selective determination of Sn 2+ in radiopharmaceutical MIBI solutions, without interference of degradation products of Sn 2+ using 1 mol L -1 HCl electrolyte.In summary, the method has advantages, such as the easy and quick preparation of the electrolyte, rapid analyses, reproducibility, and can be applied in a routine laboratory.

Figure 7 .
Figure 7. Voltammogram showing the specificity of the analysis for Sn 2+ at 1 mol L -1 HCl.For this parameter, MIBI radiopharmaceutical kits and excipients without Sn 2+ were analyzed.

Figure 9 .
Figure 9. Linearity test of the method represented by three calibration curves.

Table 1 .
Decay of the current of Sn 2+ 1, 2, 13 and 23 days after preparation of solution for reading n.d.: not determined.

Table 2 .
Intra and inter-assay accuracy and precision of the method

Table 3 .
Robustness evaluation of the method