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Rapid radiochemical purity testing for [131I]m-iodobenzylguanidine
Vol. 22,No. 2, pp. 2217229, 1995 Copyright 0 1995ElsevierScienceLtd Printedin Great Britain. All rightsreserved 0969~8051195 $9.50+ 0.00
Nucl. Med. Biol.
Pergamon
0969-8051(94)00087-S
Rapid Radiochemical Purity Testing for [‘311]m-1odobenzylguanidine J. C. HUNG* and M. E. WILSON Nuclear Medicine, Department of Diagnostic Radiology, Mayo Clinic, 200 First Street SW., Rochester, MN 55905, U.S.A. (Accepted 16 June 1994)
The standard high pressure liquid chromatography (HPLC) method for determining radiochemical purity (RCP) of [‘3’I]m -iodobenzylguanidine ([‘3’I]m IBG) is cumbersome and time-consuming. A simplified and rapid separation method with the use of a reverse-phase extraction chromatography technique (i.e. Sep-Pak” Cl8 [SPC] cartridge) is described. The new SPC system closely correlates (r = 0.99) to the standard HPLC method, and the time to complete the SPC analysis is less than 5 min.
Introduction
using an SPC cartridge for RCP analysis of [‘3’I]mIBG. In his method, the SPC cartridge is initially conditioned by elution with 5 ml ethanol followed with 5 ml H,O. The free 13’1is eluted with 4 ml H,O, and the [‘3’I]mIBG remains in the cartridge. Verbruggen’s SPC method provides a rapid RCP determination. However, the method requires that the [‘3’I]m IBG sample be alkalinized in order to obtain satisfactory separation, and [‘3’I]mIBG is retained in the cartridge which does not fulfill FDA requirements (i.e. both [‘3’I]mIBG and free 13’1should be separated and eluted from the cartridge). Ehninger et al. (1987) also published a similar method with the use of a Baker Cl8 column; however, no extensive validation data of the method are available. The SPC extraction method developed by Wafelman et al. (1992, 1993) separates and removes both [‘3’I]mIBG and free 13’1from the SPC cartridge. However, the method requires that the [“‘I]mIBG test sample be acidified initially in order to minimize [‘3’I]mIBG activity remaining in the cartridge, and multiple steps of elution (i.e. total of nine washings) are required for completing RCP analysis. The purpose of our study was to develop a rapid method for the determination of RCP values for [‘3’I]mIBG in compliance with FDA requirements.
[‘311]mIBG has been shown to be of great value in localization and treatment of metastatic pheochromocytoma and neuroblastoma (Sisson et al., 1981; Munkner, 1985). In our institution, we use a radiolabeling method developed by Mock and Weiner (1988) for the in-house preparation of [‘311]mIBG. Although [‘3’I]mIBG has been recently approved by the U.S. Food and Drug Administration (FDA), the FDA requires that quality control including RCP determination of [“‘I]mIBG be performed for any investigational use of this radiopharmaceutical. The U.S. FDA further requires an RCP value of greater than 90% during the entire shelf life of [“‘I]mIBG, that the RCP testing system provide a clear separation of [‘3’I]mIBG and free 13’1, and that both radiochemical species must migrate from the origin. Although the standard HPLC system can elute and separate both [‘3’I]mIBG and free 13’1,it is not only costly and time-consuming to operate an HPLC system, but special technical expertise is also required to maintain the HPLC system. The nuclear pharmacy of the University of Michigan Hospital does provide a thin layer chromatography system with the use of a C-18 reversed phase # KC’,F glass plate (Whatman International Ltd., Maidstone, U.K.) as the stationary phase and acetonitrile/O.Ol M dibutylammonium phosphate (80/20) as the mobile phase. The R, values for [‘3’I]mIBG and free 13’1are 0.4-0.5 and 0.9-1.0, respectively. However, it is also very timeconsuming (i.e. l-2 hr). Verbruggen (1987) developed a liquid-solid reverse-phase extraction method by
Materials Preparation
and Methods
of ['3'I]mIBG
According to the method developed by Mock and Weiner (1988) the preparation of [‘3’I]m IBG is relatively simple to perform and can produce [‘3’I]mIBG of a relatively high labeling efficiency (i.e. 84.7 f 7.0%, n = 70) and a high radiochemical purity (i.e. 96.4 + 2.9%, n = 70).
*Author for correspondence. 221
.I. c‘. Hung and M. E. Wilson
228 SmL EtOH
5mL Hfi
0.10.2
1 Dii
5mL THF: 0.1 M HzPOd (25:75, v/v)
5mL f-b0
mL
Er [l-131] mlBG
1
1 count
Discard
Free 131,
Fig. 1. Schematic representation
of [‘3’I]mIBG
RCP analysis of [‘311]mIBG with HPLC The HPLC system involved the use of a C-18 reverse phase column (4.6 mm x 15 cm) (Ultrasphere ODS 5 pm, Beckman Instruments, Inc., San Ramon, CA, U.S.A.) with a Beckman dual pump gradient chromatographic system (System Gold, Programmable Solvent Module 126). The mobile phase for the linear gradient elution system consisted of tetrahydrofuran (THF): 0.1 M NaH,PO, (25: 75, v/v). The flow rate was maintained at 1.5 ml/min. Eluent radioactivity was detected with a NaI(T1) scintillation detector (Beckman 170 Radioisotope Detector) connected to an integrator (Beckman 427 Integrator) for peak integration. In a typical run, free 13’1 had a retention time of - 1.2 min and the [‘3’I]m IBG, -2.5 min. The HPLC system was used as a standard reference method for the comparison study with the new SPC method developed in our laboratory. RCP analysis of [‘3’I]mIBG
with SPC
The new SPC system uses a reverse-phase extraction chromatography technique. As illustrated in Fig. 1, the SPC cartridge was initially activated by rinsing with 5 ml 200-proof ethanol followed by flushing the cartridge with 5 ml distilled H,O. A sample of 0.1-0.2 ml of [“‘I]mIBG solution was applied to the long end of the SPC cartridge. The cartridge was then successfully eluted with 5 ml distilled Hz0 and two 5 ml THF:O. 1 M NaH2P04 (25 : 75, v/v) solutions. The radioactivity contained in each of the three eluates and in the cartridge was measured in a dose calibrator. The RCP results obtained from the SPC system were then compared with those using the standard HPLC quality control procedure. Formulation of difSerent RCP levels of [“‘I]mIBG In order to determine the accuracy of the proposed SPC procedure, it was necessary to verify the results
1
1
Count [131,] miBG
SPC quality control procedure.
of the new SPC procedure with those of the reference HPLC procedure for various RCP levels of [‘3’I]m IBG. Sodium iodide “‘I solution was added to the [‘311]mIBG preparation in order to create the desired RCP values for comparison studies. A total of 21 test samples of [‘3’I]mIBG with RCP values ranging from 73.4 to 99.3% were prepared for the comparison study.
Results With RCP values of [‘3’I]mIBG ranging from 73.4 to 99.3% (n = 21), the percentage radioactivity of the two eluates with the 5 ml THF:O.l M NaH,PO, (25 : 75, v/v) washings correlated closely (Y = 0.99) with the percentage [‘“I]mIBG determined by the standard HPLC method. This resulted in a regression line of SPC% = 0.85(+0.03) HPLC% + 13.99 (*2.24)% with an average RCP difference of 0.3 k 1.7% (Fig. 2). There were no significant differences (P = 0.411) found in RCP level determinations ranging from 73.4 to 99.3% by both methods (Fig. 2). The total time necessary to complete the entire SPC
loo
-
QOe 0
RCP=73.4-W6.3% sPc9b=o.w(*o.o3) HPLC% + 13.W ( f 2.24)% r=o.w n-21 P-O.411 Difference-0.3*1.7%
25 so-
1
70 70
80
so
100
HPLC (U)
Fig. 2. Overall comparison between SPC and HPLC methods in the measurement of different RCP values of [‘3’I]mIBG preparations.
719
Rapid quality control method for [“31]mIBG
analysis (i.e. SPC preparation, sample applications sample washings, sample counting, and calculation) was less than 5 min. The distribution of [‘“‘I]mIBG in the second S-ml TWF:O.l M NaH,PO,
fraction comprised 2.4 + 1.3%
(n = 21) of the total [“‘I]mIBG radioactivity in the final preparation. The percentage radioactivity retained in the cartridge after the elution process was 1.3 rt 0.4 (12= 21). Only one out of the 21 [‘3’I]mIBG
There was a relativdy small fraction of [“‘I]m IBG (i.e. 2.42 1.3%, n ~21) in the second 5-ml THF: 0.1 M NaH,PO, elution. Although this small percentage of bound ~~3iI]~IBG has no effect upon the acceptance or rejection of [‘3’I]mIBG preparations demonstrating either high or low RCP values, it is definitive in the acceptance or rejection of [‘3’I]m IBG
preparations exhibiting borderline RCP values. In conclusion,
our new SPC system offers an
expedient (i.e. < 5 min) and reliable way to perform when it demonstrated an acceptable RCP value RCP testing on a [‘“‘1]mI3G preparation. The new (i.e. 90.4%) as determined by the reference HPLC SPC system precisely separates and elutes both method. However, it is significant to note that none [“I]mIBG and free IX’1 from the cartridge, thus of the [“‘I]mIBG preparations that had been rejected fulfillng U.S. FDA requirements for RCP testing on (i.e. RCP < 90% by the HPLC method) was accepted [‘3’I]mIBG. samples was rejected by the SPC method (i.e. 89.8%)
by the SPC method.
Discussion The aim of this study was to develop and validate a new SPC method for RCP measurement of [‘~~I]~-IIBG, since the standard HPLC method for RCP determination of /‘311]mIBGis cumbersome and time-consuming. Results obtained by our study indicate that the new SPC system requires no special preparation of [‘7’I]mIBG test sample (e.g. [‘3’I]naIBG sample does not need to be alkalinized or acidified). Wafelman 41 ni. (1993) studied the infiuence of pH of the eluent on the retention of radioactivity in an SPC cartridge. Their study indicates that as the pH of the eluent increases, the fraction of [‘3iI]mIBG radioactivity retained by the cartridge also increases. This may explain why, when using
Verbruggen’s method, the [13’I]mIBG activity was retained in the cartridge. According to the study by Wafelman et al. (1993), there should be 7.56 k 1.21 to 15.73I: 2.36% radioactivity remaining in the cartridge if the elution solvent is in the pH range 4.5-6.5. However, the solvent system (i.e. two 5 ml
THF:O.l M NaH,PO,, 25: 75, v/v) that we used to elute [‘3’I]mIBG
from the cartridge has a pH value
of * 5, and the cartridge retained only 1.3 5 0.4% (n = 21) of the total radioactivity sample.
of the [‘3’I]mIBG
Acknowledgements-We are grateful to Mrs Rose M. Busta and MS Vicki S. Krage for their assistance in typing the manuscript. This paper was presented at the 1993European Association of Nuclear Medicine Congress in Lausanne. Switzerland, on 14 October, 1993.
References Ehninger G., Klingebiel T., Kumbier I., Schuler U., Feine U., Treuner J. and Wailer H. D. (1987) Stability and pharma~okinetics of m-[‘3’I]iodobenzylguanidine in patients. Cancer Res. 41, 6147-6149. Mock B. II. and Weiner R. E. (I988) Simplified solid-state Labeling of [J3fI]m-~odo~n~yiguanidine. .4~@. tidier. ht. 39, 939-942. Munkner T. (1985) ‘l’I-metit-iodobenzylguanidine scintigraphy of neuroblastomas. Semin. Nucl. Med. 15, 154-160. Sisson J. C., Frager M. S., Valk T. W., Gross M. D., Swanson D. P., Wieland D. M., Tobes M. C., Beierwaltes W. H. and Thompson N. W. (1981) Scintigraphic localization of pheochromocytoma. N. Engl. J. Med. 305,1Z17. Verbruggen R. F. (1987) Fast high-yield labelling and quality control of [“‘I]- and [‘“‘IIMIBG. Appl. &d&t. ht. 38, 303-304. Wafelman A. R., Hoefnagel C. A. and Beijnen J. H. (1992) The use of SPE compared to HPLC for the determjna~ion of II-13Iliodide in infusion &ids of iI-13IlMIBG. Eur. J. Ntiil. Med. 19, 740 (abstract). . . Wafelman A. R., Hoefnagal C. A. and Beijnen J. Ii. (1993) Chromatographic determination of the radiochemical purity of [‘j’I]MIBG infusion fluids: a comparison and discussion of the chromatographic characteristics using three different techniques. Appl. Radiat. hot. 44,859 -867.
Nucl. Med. Biol.
Pergamon
0969-8051(94)00087-S
Rapid Radiochemical Purity Testing for [‘311]m-1odobenzylguanidine J. C. HUNG* and M. E. WILSON Nuclear Medicine, Department of Diagnostic Radiology, Mayo Clinic, 200 First Street SW., Rochester, MN 55905, U.S.A. (Accepted 16 June 1994)
The standard high pressure liquid chromatography (HPLC) method for determining radiochemical purity (RCP) of [‘3’I]m -iodobenzylguanidine ([‘3’I]m IBG) is cumbersome and time-consuming. A simplified and rapid separation method with the use of a reverse-phase extraction chromatography technique (i.e. Sep-Pak” Cl8 [SPC] cartridge) is described. The new SPC system closely correlates (r = 0.99) to the standard HPLC method, and the time to complete the SPC analysis is less than 5 min.
Introduction
using an SPC cartridge for RCP analysis of [‘3’I]mIBG. In his method, the SPC cartridge is initially conditioned by elution with 5 ml ethanol followed with 5 ml H,O. The free 13’1is eluted with 4 ml H,O, and the [‘3’I]mIBG remains in the cartridge. Verbruggen’s SPC method provides a rapid RCP determination. However, the method requires that the [‘3’I]m IBG sample be alkalinized in order to obtain satisfactory separation, and [‘3’I]mIBG is retained in the cartridge which does not fulfill FDA requirements (i.e. both [‘3’I]mIBG and free 13’1should be separated and eluted from the cartridge). Ehninger et al. (1987) also published a similar method with the use of a Baker Cl8 column; however, no extensive validation data of the method are available. The SPC extraction method developed by Wafelman et al. (1992, 1993) separates and removes both [‘3’I]mIBG and free 13’1from the SPC cartridge. However, the method requires that the [“‘I]mIBG test sample be acidified initially in order to minimize [‘3’I]mIBG activity remaining in the cartridge, and multiple steps of elution (i.e. total of nine washings) are required for completing RCP analysis. The purpose of our study was to develop a rapid method for the determination of RCP values for [‘3’I]mIBG in compliance with FDA requirements.
[‘311]mIBG has been shown to be of great value in localization and treatment of metastatic pheochromocytoma and neuroblastoma (Sisson et al., 1981; Munkner, 1985). In our institution, we use a radiolabeling method developed by Mock and Weiner (1988) for the in-house preparation of [‘311]mIBG. Although [‘3’I]mIBG has been recently approved by the U.S. Food and Drug Administration (FDA), the FDA requires that quality control including RCP determination of [“‘I]mIBG be performed for any investigational use of this radiopharmaceutical. The U.S. FDA further requires an RCP value of greater than 90% during the entire shelf life of [“‘I]mIBG, that the RCP testing system provide a clear separation of [‘3’I]mIBG and free 13’1, and that both radiochemical species must migrate from the origin. Although the standard HPLC system can elute and separate both [‘3’I]mIBG and free 13’1,it is not only costly and time-consuming to operate an HPLC system, but special technical expertise is also required to maintain the HPLC system. The nuclear pharmacy of the University of Michigan Hospital does provide a thin layer chromatography system with the use of a C-18 reversed phase # KC’,F glass plate (Whatman International Ltd., Maidstone, U.K.) as the stationary phase and acetonitrile/O.Ol M dibutylammonium phosphate (80/20) as the mobile phase. The R, values for [‘3’I]mIBG and free 13’1are 0.4-0.5 and 0.9-1.0, respectively. However, it is also very timeconsuming (i.e. l-2 hr). Verbruggen (1987) developed a liquid-solid reverse-phase extraction method by
Materials Preparation
and Methods
of ['3'I]mIBG
According to the method developed by Mock and Weiner (1988) the preparation of [‘3’I]m IBG is relatively simple to perform and can produce [‘3’I]mIBG of a relatively high labeling efficiency (i.e. 84.7 f 7.0%, n = 70) and a high radiochemical purity (i.e. 96.4 + 2.9%, n = 70).
*Author for correspondence. 221
.I. c‘. Hung and M. E. Wilson
228 SmL EtOH
5mL Hfi
0.10.2
1 Dii
5mL THF: 0.1 M HzPOd (25:75, v/v)
5mL f-b0
mL
Er [l-131] mlBG
1
1 count
Discard
Free 131,
Fig. 1. Schematic representation
of [‘3’I]mIBG
RCP analysis of [‘311]mIBG with HPLC The HPLC system involved the use of a C-18 reverse phase column (4.6 mm x 15 cm) (Ultrasphere ODS 5 pm, Beckman Instruments, Inc., San Ramon, CA, U.S.A.) with a Beckman dual pump gradient chromatographic system (System Gold, Programmable Solvent Module 126). The mobile phase for the linear gradient elution system consisted of tetrahydrofuran (THF): 0.1 M NaH,PO, (25: 75, v/v). The flow rate was maintained at 1.5 ml/min. Eluent radioactivity was detected with a NaI(T1) scintillation detector (Beckman 170 Radioisotope Detector) connected to an integrator (Beckman 427 Integrator) for peak integration. In a typical run, free 13’1 had a retention time of - 1.2 min and the [‘3’I]m IBG, -2.5 min. The HPLC system was used as a standard reference method for the comparison study with the new SPC method developed in our laboratory. RCP analysis of [‘3’I]mIBG
with SPC
The new SPC system uses a reverse-phase extraction chromatography technique. As illustrated in Fig. 1, the SPC cartridge was initially activated by rinsing with 5 ml 200-proof ethanol followed by flushing the cartridge with 5 ml distilled H,O. A sample of 0.1-0.2 ml of [“‘I]mIBG solution was applied to the long end of the SPC cartridge. The cartridge was then successfully eluted with 5 ml distilled Hz0 and two 5 ml THF:O. 1 M NaH2P04 (25 : 75, v/v) solutions. The radioactivity contained in each of the three eluates and in the cartridge was measured in a dose calibrator. The RCP results obtained from the SPC system were then compared with those using the standard HPLC quality control procedure. Formulation of difSerent RCP levels of [“‘I]mIBG In order to determine the accuracy of the proposed SPC procedure, it was necessary to verify the results
1
1
Count [131,] miBG
SPC quality control procedure.
of the new SPC procedure with those of the reference HPLC procedure for various RCP levels of [‘3’I]m IBG. Sodium iodide “‘I solution was added to the [‘311]mIBG preparation in order to create the desired RCP values for comparison studies. A total of 21 test samples of [‘3’I]mIBG with RCP values ranging from 73.4 to 99.3% were prepared for the comparison study.
Results With RCP values of [‘3’I]mIBG ranging from 73.4 to 99.3% (n = 21), the percentage radioactivity of the two eluates with the 5 ml THF:O.l M NaH,PO, (25 : 75, v/v) washings correlated closely (Y = 0.99) with the percentage [‘“I]mIBG determined by the standard HPLC method. This resulted in a regression line of SPC% = 0.85(+0.03) HPLC% + 13.99 (*2.24)% with an average RCP difference of 0.3 k 1.7% (Fig. 2). There were no significant differences (P = 0.411) found in RCP level determinations ranging from 73.4 to 99.3% by both methods (Fig. 2). The total time necessary to complete the entire SPC
loo
-
QOe 0
RCP=73.4-W6.3% sPc9b=o.w(*o.o3) HPLC% + 13.W ( f 2.24)% r=o.w n-21 P-O.411 Difference-0.3*1.7%
25 so-
1
70 70
80
so
100
HPLC (U)
Fig. 2. Overall comparison between SPC and HPLC methods in the measurement of different RCP values of [‘3’I]mIBG preparations.
719
Rapid quality control method for [“31]mIBG
analysis (i.e. SPC preparation, sample applications sample washings, sample counting, and calculation) was less than 5 min. The distribution of [‘“‘I]mIBG in the second S-ml TWF:O.l M NaH,PO,
fraction comprised 2.4 + 1.3%
(n = 21) of the total [“‘I]mIBG radioactivity in the final preparation. The percentage radioactivity retained in the cartridge after the elution process was 1.3 rt 0.4 (12= 21). Only one out of the 21 [‘3’I]mIBG
There was a relativdy small fraction of [“‘I]m IBG (i.e. 2.42 1.3%, n ~21) in the second 5-ml THF: 0.1 M NaH,PO, elution. Although this small percentage of bound ~~3iI]~IBG has no effect upon the acceptance or rejection of [‘3’I]mIBG preparations demonstrating either high or low RCP values, it is definitive in the acceptance or rejection of [‘3’I]m IBG
preparations exhibiting borderline RCP values. In conclusion,
our new SPC system offers an
expedient (i.e. < 5 min) and reliable way to perform when it demonstrated an acceptable RCP value RCP testing on a [‘“‘1]mI3G preparation. The new (i.e. 90.4%) as determined by the reference HPLC SPC system precisely separates and elutes both method. However, it is significant to note that none [“I]mIBG and free IX’1 from the cartridge, thus of the [“‘I]mIBG preparations that had been rejected fulfillng U.S. FDA requirements for RCP testing on (i.e. RCP < 90% by the HPLC method) was accepted [‘3’I]mIBG. samples was rejected by the SPC method (i.e. 89.8%)
by the SPC method.
Discussion The aim of this study was to develop and validate a new SPC method for RCP measurement of [‘~~I]~-IIBG, since the standard HPLC method for RCP determination of /‘311]mIBGis cumbersome and time-consuming. Results obtained by our study indicate that the new SPC system requires no special preparation of [‘7’I]mIBG test sample (e.g. [‘3’I]naIBG sample does not need to be alkalinized or acidified). Wafelman 41 ni. (1993) studied the infiuence of pH of the eluent on the retention of radioactivity in an SPC cartridge. Their study indicates that as the pH of the eluent increases, the fraction of [‘3iI]mIBG radioactivity retained by the cartridge also increases. This may explain why, when using
Verbruggen’s method, the [13’I]mIBG activity was retained in the cartridge. According to the study by Wafelman et al. (1993), there should be 7.56 k 1.21 to 15.73I: 2.36% radioactivity remaining in the cartridge if the elution solvent is in the pH range 4.5-6.5. However, the solvent system (i.e. two 5 ml
THF:O.l M NaH,PO,, 25: 75, v/v) that we used to elute [‘3’I]mIBG
from the cartridge has a pH value
of * 5, and the cartridge retained only 1.3 5 0.4% (n = 21) of the total radioactivity sample.
of the [‘3’I]mIBG
Acknowledgements-We are grateful to Mrs Rose M. Busta and MS Vicki S. Krage for their assistance in typing the manuscript. This paper was presented at the 1993European Association of Nuclear Medicine Congress in Lausanne. Switzerland, on 14 October, 1993.
References Ehninger G., Klingebiel T., Kumbier I., Schuler U., Feine U., Treuner J. and Wailer H. D. (1987) Stability and pharma~okinetics of m-[‘3’I]iodobenzylguanidine in patients. Cancer Res. 41, 6147-6149. Mock B. II. and Weiner R. E. (I988) Simplified solid-state Labeling of [J3fI]m-~odo~n~yiguanidine. .4~@. tidier. ht. 39, 939-942. Munkner T. (1985) ‘l’I-metit-iodobenzylguanidine scintigraphy of neuroblastomas. Semin. Nucl. Med. 15, 154-160. Sisson J. C., Frager M. S., Valk T. W., Gross M. D., Swanson D. P., Wieland D. M., Tobes M. C., Beierwaltes W. H. and Thompson N. W. (1981) Scintigraphic localization of pheochromocytoma. N. Engl. J. Med. 305,1Z17. Verbruggen R. F. (1987) Fast high-yield labelling and quality control of [“‘I]- and [‘“‘IIMIBG. Appl. &d&t. ht. 38, 303-304. Wafelman A. R., Hoefnagel C. A. and Beijnen J. H. (1992) The use of SPE compared to HPLC for the determjna~ion of II-13Iliodide in infusion &ids of iI-13IlMIBG. Eur. J. Ntiil. Med. 19, 740 (abstract). . . Wafelman A. R., Hoefnagal C. A. and Beijnen J. Ii. (1993) Chromatographic determination of the radiochemical purity of [‘j’I]MIBG infusion fluids: a comparison and discussion of the chromatographic characteristics using three different techniques. Appl. Radiat. hot. 44,859 -867.