- Email: [email protected]
Validation of an alternative radiochemical purity method for [99mTc]pentetate ([99mTc]DTPA)
Applied Radiation and Isotopes 82 (2013) 322–324
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Technical note
Validation of an alternative radiochemical purity method for [99mTc]pentetate ([99mTc]DTPA) María Candela Borré n, Fiorella C. Tesán, Natalia M. Leonardi, Marcela B. Zubillaga, M. Jimena Salgueiro Radioisotope Laboratory, Physics Department, School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956 PB, Buenos Aires (C1113AAD), Argentina
H I G H L I G H T S
European Pharmacopoeia's RCP method for [99mTc]DTPA involves ITLC and is widely used. ITLC has not regained fluent availability since PALL discontinued it. We proposed an alternative method that involves paper and TLC chromatography. The alternative method proved to be specific, precise, accurate, linear and robust. The alternative method has been validated according to ICH international standards.
art ic l e i nf o
a b s t r a c t
Article history: Received 20 March 2013 Received in revised form 9 August 2013 Accepted 18 September 2013 Available online 3 October 2013
[99mTc]pentetate ([99mTc]DTPA) is the most commonly used radiopharmaceutical renography agent. The aim of this work was to validate an alternative method for assessing [99mTc]DTPA radiochemical purity (RCP), according to the ICH Q2(R1) guidance: “Validation of Analytical Procedures”. The proposed method is composed of two chromatographic systems. System A is a miniaturized system of thin layer chromatography (TLC) silica gel impregnated aluminum strips as stationary phase (SP) and distilled water as mobile phase (MP). System B consists of Whatman 1 paper strips as SP and methyl ethyl ketone as MP. Results indicate that the proposed RCP method has been validated, as it is specific, precise, accurate, linear and robust. Therefore, it can be used as an alternative method for RCP quality control purposes and as stability indicator as well. & 2013 Elsevier Ltd. All rights reserved.
Keywords: DTPA Pentetate Radiochemical purity ITLC Validation 99m Tc
1. Introduction Radiochemical purity (RCP) is an important quality parameter for radiopharmaceuticals, as their radiochemical form determines their biodistribution (Vallabhajosula et al., 2010). Several pharmacopoeias contain RCP analytical methods for most radiopharmaceuticals. [99mTc]pentetate ([99mTc]DTPA) is the most commonly used radiopharmaceutical renography agent. The [99mTc]DTPA RCP method described in the United States Pharmacopeia consists of an electrophoretic technique that is not practical to perform routinely in Nuclear Medicine Centers (USP 32-NF 27, 2009). n Corresponding author. Tel.: þ 54 11 4964 8202/8277x33; fax: þ54 11 4964 8204. E-mail addresses: [email protected], [email protected] (M.C. Borré).
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.09.007
European Pharmacopoeia's chromatographic method is widely used as it is fast and easy. This method involves instant thin layer chromatography (ITLC) (EP 6th Edition, 2008). After PALL corporation discontinued silica gel impregnated glass fiber sheets (ITLCSG) in 2008, its availability was compromised (Eggert et al., 2010; Henning et al., 2010; Ivanov et al., 2010; Millar et al., 2009, 2010; Ponto, 2011; Wunderlich et al., 2010). ITLC-SG availability has not been fully restored yet, at least in Argentina. This is to some extent, due to import policies and resellers' lack of interest in items that are not in high demand. Such circumstances may force NMCs to stop performing the required RCP tests for 99mTc radiopharmaceuticals, putting the quality system at risk (IAEA, 2006). In order to avoid that outcome, the aim of this work is validating, according to the ICH Q2 (R1) guidance “Validation of Analytical Procedures” (ICH, 2005), an alternative method for assessing [99mTc]DTPA RCP.
M.C. Borré et al. / Applied Radiation and Isotopes 82 (2013) 322–324
2. Methods 2.1. Radiolabeling Radiolabeling procedure of DTPA chelator (Tecnonuclear SA, Argentina) was performed according to the manufacturer instructions. Briefly, 37–740 MBq (1–20 mCi) of a pertechnetate solution eluted from a 99Mo–99mTc generator (Laboratorios Bacon SAIC, Argentina) were added to the 5.0 mg freeze-dried DTPA formulation. Saline solution was added to the preparation to a final volume of 3 ml, reaching an activity concentration of 12.3– 246 MBq/ml (0.33–6.66 mCi/ml). Each vial was shaken vigorously and then allowed to stand at room temperature for at least 10 min before its analysis. 2.2. Chromatographic studies The proposed method consists of two chromatographic systems. 2.2.1. System A (miniaturized system) An aliquot (5 mL) of [99mTc]DTPA was spotted on a 4 cm TLC silica gel aluminum strip (Merk, Germany), and it was allowed to dry. Then, the strip was placed in a saturated chamber for being developed with distilled water. In this chromatographic system reduced, and hydrolyzed 99mTc species remain at the origin (Rf ¼ 0.0–0.1) whereas free [99mTc]pertechnetate and [99mTc]DTPA are found at Rf ¼0.9–1.0. The strip was cut at Rf ¼0.5 and the radioactivity of each piece of the strip was determined. 2.2.2. System B An aliquot (5 mL) of [99mTc]DTPA was spotted on a 10 cm Whatman 1 paper strip and it was allowed to dry. Then, the strip was placed in a saturated chamber for being developed with methyl-ethyl-ketone. In this chromatographic system [99mTc]DTPA and reduced, hydrolyzed 99mTc species remain at the origin (Rf ¼ 0.0–0.1) whereas free [99mTc]pertechnetate is found at Rf ¼0.9–1.0. The strip was cut at Rf ¼0.5 and the radioactivity of each piece of paper was determined. 2.3. Measurements
323
ranged between 37 and 740 MBq/ml (1–20 mCi/ml). Results were expressed as the equation obtained from the linear regression (recovered activity as a function of spotted activity) and its correlation coefficient (r2). Accuracy was assessed by analyzing 5 radiopharmaceutical solutions having activity concentrations that ranged between 37 and 740 MBq/ml (1–20 mCi/ml). The results were expressed as the percentage of recovery of each of the proposed systems (A and B) with respect to the reference method. Precision: to evaluate precision, repeatability and intermediate precision were assayed. Repeatability was evaluated by the same operator performing 10 determinations on the same day and on the same equipment. Intermediate precision was evaluated by 2 operators performing 10 determinations on different days in different weeks. The results were compared using the Student t test. Results were expressed as coefficient of variation (CV) of the radiochemical purity percentages determinations. Robustness was evaluated by assaying spot drying, chamber saturation and SP activation. The results were expressed as CV of both systems (A and B).
3. Results and discussion Results indicate that the suggested chromatographic method for [99mTc]DTPA RCP assessment has been validated according to ICH international standards. Analyzed characteristics proved that the studied method is specific, precise, accurate, linear and robust. Evaluation of chromatographic profiles showed similar patterns between the proposed and the reference method, proving that the first one is as specific as the second one (data not shown). In addition, it proved to be linear, as the result from the statistical test suggested that the deviation from linearity for each chromatographic system was not significant (P4 0.05; Fig. 1). Accuracy assessment showed percentages of recovery that ranged between 93.6% and 99.7% (mean ¼97.8 and CV ¼0.03%) for system A, and from 99.9% to 101.6% (mean ¼100.3 and CV ¼0.007%) for system B, being similar to the ones that are available in bibliography (Calmanovici et al., 2005; El-Moghazy et al., 2009). In the repeatability studies the mean RCP was 97.35% and CV was 1.03%. Intermediate precision results showed that the CVs obtained by the 2 operators were not statistically different (RCP ¼ 97.35% and 96.93%, CV¼ 1.03% and 0.92%, and P 40.05). The low CV values obtained are in concordance with the ones reported for this kind
Activity was measured in an ionization chamber (VEXCAL AV-02, Veccsa SA, Argentina). Radiochemical purity samples were measured in a monochannel gamma spectrometer with a NaI(Tl) standard well crystal (Alfanuclear SAIC, Argentina), which was previously set at optimal conditions. All counting measurements were performed with a relative error lower than 1%. 2.4. Validation As RCP is a quantitative test for measuring impurities, the following parameters were assessed: specificity, linearity, range, accuracy, precision, quantitation limit and robustness. All of the above mentioned validation characteristics were evaluated by comparing with the European Pharmacopoeia's method as reference, which consists of 2 chromatographies that involve ITLC-SG as SP: one with saline solution and the other with methyl-ethyl-ketone as MP (EP 6th Edition, 2008). Specificity: previously mentioned impurities were simulated with free [99mTc]pertechnetate eluted from a 99 Mo–99mTc generator and a colloid consisting of [99mTc]stannous chloride. Impure samples were analyzed with both methods (the reference and the proposed one) comparing chromatographic profiles of each simulated impurity. Linearity was assessed by analyzing 5 radiopharmaceutical solutions having activity concentrations that
ystem
ystem
r2
0.9992
1.000
y intercept
-0.004797 ± 0.007506
-0.0001735 ± 0.0005819
Slope
0.9878 ± 0.0076
0.9967 ± 0.0007
Absolute sum of squares
0.004446
1.977 . 10-5
Fig. 1. Linearity. Linear regression of A and B chromatographic systems.
324
M.C. Borré et al. / Applied Radiation and Isotopes 82 (2013) 322–324
of chromatographic methods (AOAC/FAO/IAEA/IUPAC Expert Consultation, 2000; Bozkurt et al., 2009). Robustness assessment confirmed that chamber saturation and SP activation do not affect the result of the assay, whereas, limiting spot drying resulted in an increase of dispersion (CV 425%), thus revealing the importance of this step. The range was not determined as the kind of equipment and RCP methodology used imply great versatility in the radiation measurements that can be accomplished. Therefore, the interval will vary according to the equipment and the selected measurement conditions, as different geometric and electronic measurement settings change the efficiency of counting determinations (Leonardi et al., 2012). The detection limit was not determined as, in routine practices, samples are measured (at any given measurement condition) for an extent of time enough to register at least 10,000 counts per minute (CV ¼ 1%). Thus, the detection limit will be very low and will vary according to the measurement conditions. Given that detection limits in radioactivity measurements are so low, it is senseless to determine the quantitation limit (Hoogerbugge and Van Zoonen, 2000; Leonardi et al., 2012). It is worth mentioning that chromatographies on ITLC-SG are performed in short times (8–10 min), while the proposed chromatographic systems are indeed slower (15–20 min). Nevertheless, the speed of the alternative method is perfectly compatible with routine nuclear medicine practices.
4. Conclusions Results indicate that the suggested chromatographic method for [99mTc]DTPA RCP assessment has been validated according to ICH international standards. Analyzed characteristics proved that the studied method is specific, precise, accurate, linear and robust. Therefore, it could be used as an alternative method for radiochemical quality control purposes and as stability indicator as well.
Acknowledgments This work was financially supported by the University of Buenos Aires UBACYT 20020100300071 Project. References AOAC/FAO/IAEA/IUPAC Expert Consultation, 2000. Guidelines for single-laboratory validation of analytical methods for trace-level concentrations of organic
chemicals. In: Fajgelj, A., Ambrus, A. (Eds.), Principles and Practices of Method Validation. Royal Society of Chemistry, Cambridge, pp. 179–252. Bozkurt, M.F., Kiratli, P.O., Konyali, D., Metin, F., 2009. Quality control of instant kit 99m Tc-mercapto acetyl triglycine with inter- and intra-operator measurements. Hellenic Journal of Nuclear Medicine 12 (1), 59–62. Calmanovici, G.P., Salgueiro, M.J., Leonardi, N.M., Goldman, C.G., Nicolini, J.O., Boccio, J.R., Caro, R.A., Zubata, P.D., Zubillaga, M.B., 2005. Quality control validation for exogenous natural surfactant labeled with 99mTc. Journal of Nuclear Medicine Technology 33, 234–237. EDQM, 2008. European Pharmacopoeia, 6th ed. Strasbourg p. 1035. Eggert, L.A., Dick, M.D., Mahoney, D.W., Olson, J.P., Werner, G.L., Hung, J.C., 2010. A rapid radiochemical purity testing method for 99mTc-tetrofosmin. Journal of Nuclear Medicine Technology 38, 81–84. El-Moghazy, S.M., El-Azem Mohamed, M.A., Mohamedb, M.F., Youssef, N.F., 2009. Development and validation of HPLC, TLC and derivative spectrophotometric methods for the analysis of ezetimibe in the presence of alkaline induced degradation products. Journal of the Chinese Chemical Society 56 (2), 360–367. Henning, T., Cutrera, P., Zimmer, M., McDonald, N., Spies, S., 2010. Non-ITLC SG miniaturized chromatography procedures to evaluate hydrolyzed reduced 99m Tc in specific 99mTc radiopharmaceuticals. Journal of Nuclear Medicine 51 (2), 2108. Hoogerbugge, R., Van Zoonen, P., 2000. Validation of analytical data in research and development environment. In: Fajgelj, A., Ambrus, A. (Eds.), Principles of Method Validation. Royal Society of Chemistry, Cambridge, pp. 19–29. IAEA, 2006. Quality assurance for radioactivity measurement in nuclear medicine. Technical reports series no. 454, Vienna. ICH, 2005. ICH harmonized tripartite guideline validation of analytical procedures: text and methodology Q2 (R1). International conference on the harmonization of technical requirements for the registration of pharmaceuticals for human use, Geneva. Ivanov, Z., Guilfoyle, R., Bokor, I., 2010. Alternatives to instant thin layer chromatography-silica gel (ITLC-SG) for use in the radiochemical purity measurement of reconstituted ‘Cold Kit’ 99mTc radiopharmaceuticals. ANZ Nuclear Medicine 41 (1), 6–8. Leonardi, N.M., Casale, G.A., Nicolini, J., Zubata, P.D., Salgueiro, M.J., Zubillaga, M.B., 2012. Validation of a paper chromatographic methodology as an alternative for determination of the radiochemical purity of Na18F. Journal of Nuclear Medicine Technology 40, 271–274. Millar, A.M., Beattie, L.A., Craig, F., O'Brien, L.M., 2009. An evaluation of GMCP-SA as a replacement for ITLC-SG when measuring the levels of radiochemical impurities in 99mTc radiopharmaceuticals by thin-layer chromatography. Journal of Labelled Compounds and Radiopharmaceuticals 52, 538–542. Millar, A.M., O'Brien, L.M., Beattie, L.A., Craig, F., 2010. An evaluation of paper chromatography for measuring the levels of radiochemical impurities in 99mTc medronate injection. Journal of Labelled Compounds and Radiopharmaceuticals 53, 11–14. Ponto, J.A., 2011. Evaluation of alternative methods for radiochemical purity testing of indium-111 capromab pendetide. Journal of the American Pharmacists Association 51 (2), 359–362. United States Pharmacopeial Convention, 2009. USP 32-NF 27, Rockville, MD, pp. 3668–3669. Vallabhajosula, S., Killeen, R.P., Osborne, J.R., 2010. Altered biodistribution of radiopharmaceuticals: role of radiochemical/pharmaceutical purity, physiological, and pharmacologic factors. Seminars in Nuclear Medicine 40 (4), 220–241. Wunderlich, G., Herrling, P., Zürn, A., Anders, P., Kotzerke, J., 2010. Chromatographic determination of radiochemical purity – replacement of ITLC SG. Nuklearmedizin 49 (2), 73–77.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Technical note
Validation of an alternative radiochemical purity method for [99mTc]pentetate ([99mTc]DTPA) María Candela Borré n, Fiorella C. Tesán, Natalia M. Leonardi, Marcela B. Zubillaga, M. Jimena Salgueiro Radioisotope Laboratory, Physics Department, School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956 PB, Buenos Aires (C1113AAD), Argentina
H I G H L I G H T S
European Pharmacopoeia's RCP method for [99mTc]DTPA involves ITLC and is widely used. ITLC has not regained fluent availability since PALL discontinued it. We proposed an alternative method that involves paper and TLC chromatography. The alternative method proved to be specific, precise, accurate, linear and robust. The alternative method has been validated according to ICH international standards.
art ic l e i nf o
a b s t r a c t
Article history: Received 20 March 2013 Received in revised form 9 August 2013 Accepted 18 September 2013 Available online 3 October 2013
[99mTc]pentetate ([99mTc]DTPA) is the most commonly used radiopharmaceutical renography agent. The aim of this work was to validate an alternative method for assessing [99mTc]DTPA radiochemical purity (RCP), according to the ICH Q2(R1) guidance: “Validation of Analytical Procedures”. The proposed method is composed of two chromatographic systems. System A is a miniaturized system of thin layer chromatography (TLC) silica gel impregnated aluminum strips as stationary phase (SP) and distilled water as mobile phase (MP). System B consists of Whatman 1 paper strips as SP and methyl ethyl ketone as MP. Results indicate that the proposed RCP method has been validated, as it is specific, precise, accurate, linear and robust. Therefore, it can be used as an alternative method for RCP quality control purposes and as stability indicator as well. & 2013 Elsevier Ltd. All rights reserved.
Keywords: DTPA Pentetate Radiochemical purity ITLC Validation 99m Tc
1. Introduction Radiochemical purity (RCP) is an important quality parameter for radiopharmaceuticals, as their radiochemical form determines their biodistribution (Vallabhajosula et al., 2010). Several pharmacopoeias contain RCP analytical methods for most radiopharmaceuticals. [99mTc]pentetate ([99mTc]DTPA) is the most commonly used radiopharmaceutical renography agent. The [99mTc]DTPA RCP method described in the United States Pharmacopeia consists of an electrophoretic technique that is not practical to perform routinely in Nuclear Medicine Centers (USP 32-NF 27, 2009). n Corresponding author. Tel.: þ 54 11 4964 8202/8277x33; fax: þ54 11 4964 8204. E-mail addresses: [email protected], [email protected] (M.C. Borré).
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.09.007
European Pharmacopoeia's chromatographic method is widely used as it is fast and easy. This method involves instant thin layer chromatography (ITLC) (EP 6th Edition, 2008). After PALL corporation discontinued silica gel impregnated glass fiber sheets (ITLCSG) in 2008, its availability was compromised (Eggert et al., 2010; Henning et al., 2010; Ivanov et al., 2010; Millar et al., 2009, 2010; Ponto, 2011; Wunderlich et al., 2010). ITLC-SG availability has not been fully restored yet, at least in Argentina. This is to some extent, due to import policies and resellers' lack of interest in items that are not in high demand. Such circumstances may force NMCs to stop performing the required RCP tests for 99mTc radiopharmaceuticals, putting the quality system at risk (IAEA, 2006). In order to avoid that outcome, the aim of this work is validating, according to the ICH Q2 (R1) guidance “Validation of Analytical Procedures” (ICH, 2005), an alternative method for assessing [99mTc]DTPA RCP.
M.C. Borré et al. / Applied Radiation and Isotopes 82 (2013) 322–324
2. Methods 2.1. Radiolabeling Radiolabeling procedure of DTPA chelator (Tecnonuclear SA, Argentina) was performed according to the manufacturer instructions. Briefly, 37–740 MBq (1–20 mCi) of a pertechnetate solution eluted from a 99Mo–99mTc generator (Laboratorios Bacon SAIC, Argentina) were added to the 5.0 mg freeze-dried DTPA formulation. Saline solution was added to the preparation to a final volume of 3 ml, reaching an activity concentration of 12.3– 246 MBq/ml (0.33–6.66 mCi/ml). Each vial was shaken vigorously and then allowed to stand at room temperature for at least 10 min before its analysis. 2.2. Chromatographic studies The proposed method consists of two chromatographic systems. 2.2.1. System A (miniaturized system) An aliquot (5 mL) of [99mTc]DTPA was spotted on a 4 cm TLC silica gel aluminum strip (Merk, Germany), and it was allowed to dry. Then, the strip was placed in a saturated chamber for being developed with distilled water. In this chromatographic system reduced, and hydrolyzed 99mTc species remain at the origin (Rf ¼ 0.0–0.1) whereas free [99mTc]pertechnetate and [99mTc]DTPA are found at Rf ¼0.9–1.0. The strip was cut at Rf ¼0.5 and the radioactivity of each piece of the strip was determined. 2.2.2. System B An aliquot (5 mL) of [99mTc]DTPA was spotted on a 10 cm Whatman 1 paper strip and it was allowed to dry. Then, the strip was placed in a saturated chamber for being developed with methyl-ethyl-ketone. In this chromatographic system [99mTc]DTPA and reduced, hydrolyzed 99mTc species remain at the origin (Rf ¼ 0.0–0.1) whereas free [99mTc]pertechnetate is found at Rf ¼0.9–1.0. The strip was cut at Rf ¼0.5 and the radioactivity of each piece of paper was determined. 2.3. Measurements
323
ranged between 37 and 740 MBq/ml (1–20 mCi/ml). Results were expressed as the equation obtained from the linear regression (recovered activity as a function of spotted activity) and its correlation coefficient (r2). Accuracy was assessed by analyzing 5 radiopharmaceutical solutions having activity concentrations that ranged between 37 and 740 MBq/ml (1–20 mCi/ml). The results were expressed as the percentage of recovery of each of the proposed systems (A and B) with respect to the reference method. Precision: to evaluate precision, repeatability and intermediate precision were assayed. Repeatability was evaluated by the same operator performing 10 determinations on the same day and on the same equipment. Intermediate precision was evaluated by 2 operators performing 10 determinations on different days in different weeks. The results were compared using the Student t test. Results were expressed as coefficient of variation (CV) of the radiochemical purity percentages determinations. Robustness was evaluated by assaying spot drying, chamber saturation and SP activation. The results were expressed as CV of both systems (A and B).
3. Results and discussion Results indicate that the suggested chromatographic method for [99mTc]DTPA RCP assessment has been validated according to ICH international standards. Analyzed characteristics proved that the studied method is specific, precise, accurate, linear and robust. Evaluation of chromatographic profiles showed similar patterns between the proposed and the reference method, proving that the first one is as specific as the second one (data not shown). In addition, it proved to be linear, as the result from the statistical test suggested that the deviation from linearity for each chromatographic system was not significant (P4 0.05; Fig. 1). Accuracy assessment showed percentages of recovery that ranged between 93.6% and 99.7% (mean ¼97.8 and CV ¼0.03%) for system A, and from 99.9% to 101.6% (mean ¼100.3 and CV ¼0.007%) for system B, being similar to the ones that are available in bibliography (Calmanovici et al., 2005; El-Moghazy et al., 2009). In the repeatability studies the mean RCP was 97.35% and CV was 1.03%. Intermediate precision results showed that the CVs obtained by the 2 operators were not statistically different (RCP ¼ 97.35% and 96.93%, CV¼ 1.03% and 0.92%, and P 40.05). The low CV values obtained are in concordance with the ones reported for this kind
Activity was measured in an ionization chamber (VEXCAL AV-02, Veccsa SA, Argentina). Radiochemical purity samples were measured in a monochannel gamma spectrometer with a NaI(Tl) standard well crystal (Alfanuclear SAIC, Argentina), which was previously set at optimal conditions. All counting measurements were performed with a relative error lower than 1%. 2.4. Validation As RCP is a quantitative test for measuring impurities, the following parameters were assessed: specificity, linearity, range, accuracy, precision, quantitation limit and robustness. All of the above mentioned validation characteristics were evaluated by comparing with the European Pharmacopoeia's method as reference, which consists of 2 chromatographies that involve ITLC-SG as SP: one with saline solution and the other with methyl-ethyl-ketone as MP (EP 6th Edition, 2008). Specificity: previously mentioned impurities were simulated with free [99mTc]pertechnetate eluted from a 99 Mo–99mTc generator and a colloid consisting of [99mTc]stannous chloride. Impure samples were analyzed with both methods (the reference and the proposed one) comparing chromatographic profiles of each simulated impurity. Linearity was assessed by analyzing 5 radiopharmaceutical solutions having activity concentrations that
ystem
ystem
r2
0.9992
1.000
y intercept
-0.004797 ± 0.007506
-0.0001735 ± 0.0005819
Slope
0.9878 ± 0.0076
0.9967 ± 0.0007
Absolute sum of squares
0.004446
1.977 . 10-5
Fig. 1. Linearity. Linear regression of A and B chromatographic systems.
324
M.C. Borré et al. / Applied Radiation and Isotopes 82 (2013) 322–324
of chromatographic methods (AOAC/FAO/IAEA/IUPAC Expert Consultation, 2000; Bozkurt et al., 2009). Robustness assessment confirmed that chamber saturation and SP activation do not affect the result of the assay, whereas, limiting spot drying resulted in an increase of dispersion (CV 425%), thus revealing the importance of this step. The range was not determined as the kind of equipment and RCP methodology used imply great versatility in the radiation measurements that can be accomplished. Therefore, the interval will vary according to the equipment and the selected measurement conditions, as different geometric and electronic measurement settings change the efficiency of counting determinations (Leonardi et al., 2012). The detection limit was not determined as, in routine practices, samples are measured (at any given measurement condition) for an extent of time enough to register at least 10,000 counts per minute (CV ¼ 1%). Thus, the detection limit will be very low and will vary according to the measurement conditions. Given that detection limits in radioactivity measurements are so low, it is senseless to determine the quantitation limit (Hoogerbugge and Van Zoonen, 2000; Leonardi et al., 2012). It is worth mentioning that chromatographies on ITLC-SG are performed in short times (8–10 min), while the proposed chromatographic systems are indeed slower (15–20 min). Nevertheless, the speed of the alternative method is perfectly compatible with routine nuclear medicine practices.
4. Conclusions Results indicate that the suggested chromatographic method for [99mTc]DTPA RCP assessment has been validated according to ICH international standards. Analyzed characteristics proved that the studied method is specific, precise, accurate, linear and robust. Therefore, it could be used as an alternative method for radiochemical quality control purposes and as stability indicator as well.
Acknowledgments This work was financially supported by the University of Buenos Aires UBACYT 20020100300071 Project. References AOAC/FAO/IAEA/IUPAC Expert Consultation, 2000. Guidelines for single-laboratory validation of analytical methods for trace-level concentrations of organic
chemicals. In: Fajgelj, A., Ambrus, A. (Eds.), Principles and Practices of Method Validation. Royal Society of Chemistry, Cambridge, pp. 179–252. Bozkurt, M.F., Kiratli, P.O., Konyali, D., Metin, F., 2009. Quality control of instant kit 99m Tc-mercapto acetyl triglycine with inter- and intra-operator measurements. Hellenic Journal of Nuclear Medicine 12 (1), 59–62. Calmanovici, G.P., Salgueiro, M.J., Leonardi, N.M., Goldman, C.G., Nicolini, J.O., Boccio, J.R., Caro, R.A., Zubata, P.D., Zubillaga, M.B., 2005. Quality control validation for exogenous natural surfactant labeled with 99mTc. Journal of Nuclear Medicine Technology 33, 234–237. EDQM, 2008. European Pharmacopoeia, 6th ed. Strasbourg p. 1035. Eggert, L.A., Dick, M.D., Mahoney, D.W., Olson, J.P., Werner, G.L., Hung, J.C., 2010. A rapid radiochemical purity testing method for 99mTc-tetrofosmin. Journal of Nuclear Medicine Technology 38, 81–84. El-Moghazy, S.M., El-Azem Mohamed, M.A., Mohamedb, M.F., Youssef, N.F., 2009. Development and validation of HPLC, TLC and derivative spectrophotometric methods for the analysis of ezetimibe in the presence of alkaline induced degradation products. Journal of the Chinese Chemical Society 56 (2), 360–367. Henning, T., Cutrera, P., Zimmer, M., McDonald, N., Spies, S., 2010. Non-ITLC SG miniaturized chromatography procedures to evaluate hydrolyzed reduced 99m Tc in specific 99mTc radiopharmaceuticals. Journal of Nuclear Medicine 51 (2), 2108. Hoogerbugge, R., Van Zoonen, P., 2000. Validation of analytical data in research and development environment. In: Fajgelj, A., Ambrus, A. (Eds.), Principles of Method Validation. Royal Society of Chemistry, Cambridge, pp. 19–29. IAEA, 2006. Quality assurance for radioactivity measurement in nuclear medicine. Technical reports series no. 454, Vienna. ICH, 2005. ICH harmonized tripartite guideline validation of analytical procedures: text and methodology Q2 (R1). International conference on the harmonization of technical requirements for the registration of pharmaceuticals for human use, Geneva. Ivanov, Z., Guilfoyle, R., Bokor, I., 2010. Alternatives to instant thin layer chromatography-silica gel (ITLC-SG) for use in the radiochemical purity measurement of reconstituted ‘Cold Kit’ 99mTc radiopharmaceuticals. ANZ Nuclear Medicine 41 (1), 6–8. Leonardi, N.M., Casale, G.A., Nicolini, J., Zubata, P.D., Salgueiro, M.J., Zubillaga, M.B., 2012. Validation of a paper chromatographic methodology as an alternative for determination of the radiochemical purity of Na18F. Journal of Nuclear Medicine Technology 40, 271–274. Millar, A.M., Beattie, L.A., Craig, F., O'Brien, L.M., 2009. An evaluation of GMCP-SA as a replacement for ITLC-SG when measuring the levels of radiochemical impurities in 99mTc radiopharmaceuticals by thin-layer chromatography. Journal of Labelled Compounds and Radiopharmaceuticals 52, 538–542. Millar, A.M., O'Brien, L.M., Beattie, L.A., Craig, F., 2010. An evaluation of paper chromatography for measuring the levels of radiochemical impurities in 99mTc medronate injection. Journal of Labelled Compounds and Radiopharmaceuticals 53, 11–14. Ponto, J.A., 2011. Evaluation of alternative methods for radiochemical purity testing of indium-111 capromab pendetide. Journal of the American Pharmacists Association 51 (2), 359–362. United States Pharmacopeial Convention, 2009. USP 32-NF 27, Rockville, MD, pp. 3668–3669. Vallabhajosula, S., Killeen, R.P., Osborne, J.R., 2010. Altered biodistribution of radiopharmaceuticals: role of radiochemical/pharmaceutical purity, physiological, and pharmacologic factors. Seminars in Nuclear Medicine 40 (4), 220–241. Wunderlich, G., Herrling, P., Zürn, A., Anders, P., Kotzerke, J., 2010. Chromatographic determination of radiochemical purity – replacement of ITLC SG. Nuklearmedizin 49 (2), 73–77.