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[11C]acetate and [11C]methionine: Improved syntheses and quality control
AppL Radiat. Isot. Vol. 46, No. 3, pp. 173-175, 1995
~
Pergamon
0969-8043(94)00133-2
[nClAeetate and 111ClMethionine:Improved Syntheses and Quality Control
Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0969-8043/95 $9.50+ 0.00
require HPLC. The synthesis requires less than 30 min to produce chemical yields from HCO2 in excess of 60%.
Experimental M. S. BERRIDGE, E. H. CASSIDY and F. MIRALDI Departments of Radiology and Chemistry, Case Western Reserve University and University Hospitals of Cleveland. Cleveland, OH 44106, U.S.A. (Received 18 August 1994; in revised form 10 October 1994)
Two HC-labeled radiopharmaceuticals which are routinely used in clinical PET procedures are [llC]acetate and [tlC]methionine. In implementing published procedures for their synthesis, modifications of the procedures and chemistry were made to create a more rapid and reproducible production process for routine clinical application We report simplified methods which allow a single apparatus to be used for all llC syntheses which use methyl iodide, Grignard reagents, or acetone as labeled synthetic precursors. A reliable TLC quality control method for [llC]acetat e is also reported.
A Scanditronix MC-17 cyclotron equipped with the manufacturer's standard HC target was used. The 14N(p,~)IIc reaction with 17MeV protons was used to produce labeled carbon dioxide. Zero grade (analyzed) nitrogen gas (Gas Technics) was used as the target gas, and the target was periodically conditioned by irradiation of 2% zero grade oxygen in zero grade nitrogen. L-Homocysteine thiolactone hydrochloride was obtained from Sigma Chemical Co., and other materials were obtained from Aldrich Chemical Co. and Fisher Scientific and used without further purification except as noted. HPLC was performed using columns and conditions noted below with a Spectra-Physics SP8700 or Beckman ll0B pump system, and a 2.5 x 7 cm NaI radiation detector (Harshaw) with associated electronics and rate meter (Canberra), with (as appropriate) a Knauer Differential Refractometer (Type 731.980) or a Hewlett-Packard HP1050 multi-wavelength detector, or a Dionex pulsed electrochemical detector. Acetate
Introduction Available syntheses of [llC]acetate (Pike et al.. 1981a,b, 1984; Del Fiore et al., 1984) from a corresponding Grignard reagent rely on purification of the product by organic extraction. This requires aqueous and organic phases to be separated remotely in a shielded enclosure. Although the method has been successfully applied in several laboratories, the extraction was difficult in our existing synthetic apparatus. Even with specialized apparatus, considerable operator skill and precision was required, so the procedure was prone to error. For reliable routine production of a variety of radiopharmaceuticals in limited laboratory space it is desirable to have all ~C labeling procedures take place in a single synthetic apparatus, so a simple purification sequence which would be easily adaptable to the apparatus and to automation was desired. Further, quality control by HPLC was accurate but inconvenient. Available TLC methods did not reliably distinguish acetate from carbonate, especially when low levels of carbonate contamination were present. A reliable TLC method for quality control was needed. Methionine was another radiopharmaceutical of interest for routine use. Two procedures for the synthesis of [lIC]methionine have been reported. Comar et al. (1976), Berger et aL (1979, 1980) and Meyer et al. (1982, 1983) methylated the precursors L-homocysteine thiolactone or DL-homocysteine in the presence of proportionally large amounts of NaOH (60:1 mole ratio) to produce labeled methionine. Langstrom and Davis (Langstrom and Lundquist, 1976; Langstrom et aL, 1987; Davis et al., 1982) converted S-benzyl cysteine to the sulfide using sodium in liquid ammonia before methylation. All approaches used high pressure liquid chromatography for purification of the product. Though all of these approaches provide satisfactory results to their users, we have made minor modifications which significantly altered the routine procedure. We have adapted the simpler method of Comar to allow a simplified purification method which is faster and does not
A Grignard reagent solution (0.05 M methyl magnesium bromide in ether) was prepared by adding 0.05 g clean Mg to 100 mL ether distilled under Ar from calcium hydride. Bromomethane was bubbled into the mixture slowly for 30 min. The reaction mixture was gently heated and stirred for 3 h under At, then collected through Teflon tubing into small Ar-flushed bottles. The reagent solution could be stored at 5°C for several months. Tetrahydrofuran (THF) was freshly distilled under Ar from sodium and benzophenone before use. HPLC was performed on an Alltech 250 x 4.6 mm Adsorbosphere SAX column. The elution solvent was 0.005 M NaH2PO4 at a flow rate of 2 mL/min (R.T.: [llC]acetate, 8 rain; carbonate 3 rain). Bromide was measured on an Alltech Universal Anion column eluted with 2.5mM aqueous 2-hydroxybenzoic acid adjusted to pH 7.9 with sodium hydroxide (R.T.: 6 rain) and magnesium was measured on an Alltech Universal Cation column eluted with 3mM aqueous methanesulfonic acid (R.T.: 9 rain). Gas chromatography was used to analyze the product solvent. It was performed on a Hewlett-Packard 5890 instrument using a thermal conductivity detector and a Porapak N (5m x 3ram) column, eluted with 15 mL/min He at 175°C. Basic thin layer chromatography (TLC) silica plates were prepared by thoroughly wetting standard plates (Merck 0.25mm, 5 x 10 cm silica) by brief immersion in a saturated solution of NaOH in methanol and then drying them in air. They could then be stored indefinitely. NaOH was added to test solutions (to bring them to 0.1 N NaOH) before their application to the basic TLC plates and elution with methanol (Rf: acetate, 0.8; carbonate, 0). Figure 1 shows the schematic diagram of the apparatus used for all ltC syntheses. The 11CO2 was trapped under vacuum in a coil cooled in liquid nitrogen and then transferred to the reaction vessel with a stream of He. It passed through a trap (Tewson et aL, 1989) to remove oxides of nitrogen and was dried by a trap containing P205. It was collected in a solution of 50/t L of 50 mM Grignard solution added to 200 pL of freshly distilled dry THF. The reaction was immediately quenched by adding 100 #L ethanol and 173
Technical Note
174
the solution was evaporated to dryness at 100°C. Additional (2 x 200/~L) portions of ethanol were added and evaporated. Water (2 x 1 mL) was added to rinse the vessel, and the mixture was passed through a pre-wet silica cartridge into a second vessel. The solution was acidified with 150 # L of 1 M HC1 and He gas was bubbled through it for 1 rain, rapidly removing [llC]carbonate. The acid was neutralized with 200#L 1M NaHCO 3 (pH7.8) solution and the product filtered through a 0.22 #m sterile, sterilizing filter. Quality control was performed by adding several drops of product solution to 0.5 mL of 0.1 N NaOH which was then spotted on a basic TLC plate and eluted (10min) as described above. Methionine
HPLC for product analysis was performed using the system described above with an Alltech analytical reverse phase Econosil C-18 column (250 x 4.6mm), eluted with 0.001 M NaH2PO 4 adjusted to pH 3.0 with phosphoric acid (RT: methionine, 3 min; methanol, 2 min). The product was detected by radioactivity and by U.V. absorbance of added standard methionine at 220 rim. For routine quality control, the product pH was verified (7 < pH < 8.5) with indicator (1-10 pH) paper and purity was measured by TLC on Merck 0.25 mm, 5 × 10 cm silica plates eluted with 2% NH4OH in methanol. The radioactive product was measured on a Berthold linear TLC analyzer. Methionine carrier was added to the product or chromatographed separately and was detected with iodine vapor. The same apparatus was used for the [HC]acetate synthesis (Fig. 1)0 but sodium carbonate was substituted for silica in the trap in order to remove HI vapor. Labeled carbon dioxide was prepared as above and collected in a solution of 2 #mol LiA1H4 in 200 #L freshly distilled THF. After evaporating the THF and cooling the vessel, 400 #L of 57% HI was added (Langstrom et al., 1976). Labeled methyl iodide was distilled at 110°C into the second reaction vessel aided by a He gas flow (20 mL/min). It was collected at - 7 0 ° C in a solution of 6.5/~mol L-homocysteine thiolactone .HC1 in 100 #1 dry THF, mixed with a solution of 5 # L of 5 M aqueous NaOH (25 #mol) in 50 # L of ethanol. The mixture was refluxed for 10min at 75°C, then the temperature was increased to 150°C to evaporate the solution to dryness under He gas flow. Ethanol (2 x 200 ~tL) was added and evaporated to remove volatile materials ([llC]iodomethane, [lIC]methanol and THF). The residue
ROW
was taken up in 100 #L of 1 M HC1, neutralized with 300 #L 1 M NaHCO3 and filtered through a sterile 0.2 #m filter into a sterile syringe or vial. Two 2.0 mL portions of sterile saline (USP) were added and used to rinse residual product into the collection vessel.
Results and Discussion The change in the acetate synthesis compared to previous methods was that solvent and volatile impurities were removed by evaporation with ethanol after the Grignard reaction and before acidification. This method eliminated the need for liquid extraction, and allowed complete removal of radiochemical impurities and solvents. One evaporation of 200 #L ethanol reduced the THF content of the product solution below 0.5%, and a second evaporation left no detectable THF in the product. No labeled acetate (CH3 ltCOO - + MgBr) was lost during the procudure. Most of the salts created as reaction byproducts were removed after acidification by filtration through a bed of silica. Traces of Br- and Mg +÷ remained, but in amounts (2-20 #g) that are negligible from the standpoint of toxicity or pharmacological effects (USDA recommended daily allowance for Mg is 300 mg). The use of separate columns to specifically absorb Mg and bromide was reported by Meyer et al. (1993) in a conceptually similar system. In addition to these additional columns, that system also employed phosphoric acid, a longer heating cycle for CO2 removal, and HPLC for quality control. [llC]Carbonate was easily removed after acidification of the mixture, without significant loss of labeled acetate. The purification procedure was therefore effective, and easily manageable by a remotely controlled or automated system. HPLC systems as previously described by others can be used if desired to remove the traces of salt remaining in the product, but are not necessary to achieve a satisfactory injectable material. A simple thin layer chromatography (TLC) procedure was developed to detect any [HC]-carbonate, the only potential radiochemical impurity. Measurement of trace carbonate impurity was initially difficult due to its volatility under acid conditions and to the similarity of its chromatographic properties with those of acetate. This problem was solved by using strongly basic silica TLC plates. Tests of the procedure using standardized mixtures of labeled carbonate and acetate showed that the method was accurate, and that the limit of detectability was well under 0.1%. With the standard
terrors
Met~ CarlTIdge
~-~
Fig. 1. Synthetic apparatus used to prepare all HC-labeled products. The HPLC apparatus shown is optional for this work, though it is used in other preparations.
Technical Note
9O, 80 70.
o~,
60.
~" 4o
10 0
, 2
.
, 4
.
t
.
6
, 8
'
,
•
10
, 12
Mote Ratio: N a O H / H o m o ~ s t e i n e Thiolactone
Fig. 2. Yield of methionine as a function of the mole ratio of sodium hydroxide to homocysteine thiolactone. procedure, the amount of labeled carbonate present was never above 2%, with a normal range of 0.04-1.2%. The procedure gave sterile, pyrogen-free, isotonic and radiochemically pure [HC]-acetate in 60% chemical yield (based on I1CO2) within 15min from the end of bombardment. Typically, an irradiation of 30 #A for 30rain produced 1700 mCi of HC at the end of bombardment and 600 mCi of acetate and end of synthesis. The significant improvement in the methionine process was the marked reduction in the quantity of sodium hydroxide in the reaction mix compared to published methods. The reduction was achieved by reducing the quantity of homocysteine thiolactone as well as the NaOH stoichiometry (4: 1 mole ratio NaOH:thiolactone). This increased the yield of methionine with respect to methyl iodide and, more importantly, it allowed us to simplify the purification process. Figure 2 shows the relationship of the NaOH/thiolactone ratio to the chemical yield of the reaction. Reduced Na content allowed us to produce an isotonic solution without an HPLC purification, and this saved valuable time and simplified the synthetic apparatus while maintaining the final radiochemical purity. In this procedure, all radioactive and undesirable organic impurities were removed by evaporation. A trace of the synthetic precursor, L-homocysteine thiolactone, which could have been removed by HPLC, was allowed to remain in the product. L-homocysteine thio. lactone is a normal metabolite which is present in human serum (1.67#mol/mL; McKully and Vezeridis, 1988) in a concentration similar to that of the injectable product (1.5#mol/mL). The maximum quantity which could be injected in this product (6.5 ~mol) is negligible in comparison to the quantity normally present in the blood pool, and therefore there is no need to remove it. References
Berger G., Maziere M., Knipper R., Prenant C. and Comar D. (1979) Automated synthesis of HC-labeled 8
175
radiopharmaceuticals: imipramine, chlorpromazine, nicotine and methionine. Int. J. Appl. Radiat. Isot. 30, 393. Berger G., Maziere M., Godot J. M., Prenant C. and Comar D. (1980) HPLC Purification of l~C-labeled radiopharmaceuticals. Int. J. Appl. Radiat. Isot. 31, 641. Comar D., Cartron J.-C., Maziere M. and Marazano C. (1976) Labeling and metabolism of methionine-methyl]1C. Eur. J, Nucl. Med. l, 11. Davis J., Yano Y,, Cahoon J. and Budinger T. F. (1982) Preparation of HC-methyl iodide and L-[S-methyl-llC] methionine by an automated continuous flow process. Int. J. AppL Radiat. Isot. 33, 363. Del Fiore G., Peters J. M., Quaglia L., Piette J. L., Cantineau R., De Landsheere C., Raets D. and Rigo P. (1984) Automated production of carbon-11 labeled acetate to allow detection of transient myocardial ischemia in man, using positron emission tomography. J. Radioanal. Nuel. Chem. 87, 1-14. Langstrom B. and Lundquist H. (1976) The preparation of ltC-methyl iodide and its use in the synthesis of HC-methyl-L-methionine. Int. J. Appl. Radiat. Isot. 27, 357. Langstrom B., Antoni G,, Gullberg P., Halldin C., Malmborg P., Nagrcn K., Rimland A. and Svard H. (1987) Synthesis of L- and D-[methyl-~lC] methionine. J. Nucl. Med. 28, 1037. McCully K. S. and Vezeridis M. P. (1988) Homocysteine thiolactone in arteriosclerosis and cancer. Res. Comm. Chem. Path. Pharm. 59, 107. Meyer G.-J., Osterholz A. and Hundeshagen H. (1982) Routine production and quality control of HC-L-methionine. J. Lab. Comp. Radiopharm. 19, 1286. Meyer G.-J., Osterholz A. and Hundeshagen H. (1983) Routine quality control of ~C-labeled radiopharmaceuticals by high pressure liquid chromatography. J. RadioanaL Chem. 80, 229. Meyer G.-J., Gfinther K., Matzke K.-H., Harms Th. and Hundeshagen H. (1993) A modified preparation for llC acetate, preventing liquid phase extraction steps. J. Lab. Comput. Radiopharm. 32, 182-183 (abstract). Pike V. W., Eakins M. N., Allan R. M. and Selwyn A. P. (1981a) Preparation of [1-~lC]acetate--an agent for the study of myocardial metabolism by positron emission tomography. Int. J. Appl. Radiat. Isot. 30, 505. Pike V. W., Eakins M. N., Allan R. M. and Selwyn A. P. (1981b) Preparation of carbon-ll labeled acetate and palmitic acid for the study of myocardial metabolism by emission--computerized axial tomography. J. Radioanal. Chem. 64, 291. Pike V. W., Horlock P. L., Brown C. and Clark J. C. (1984) The remotely-controlled preparation of a ~]C-labeled radiopharmaceutical[1-HC]acetate. Int. J. Appl. Radiat. Isot. 35, 623. Tewson T. J., Banks W., Franceschini M. P. and Hoffpavir J. (1989) A trap for the removal of oxides of nitrogen from ]1C-carbon dioxide. AppL Radiat. Isot. 40, 765-768.
~
Pergamon
0969-8043(94)00133-2
[nClAeetate and 111ClMethionine:Improved Syntheses and Quality Control
Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0969-8043/95 $9.50+ 0.00
require HPLC. The synthesis requires less than 30 min to produce chemical yields from HCO2 in excess of 60%.
Experimental M. S. BERRIDGE, E. H. CASSIDY and F. MIRALDI Departments of Radiology and Chemistry, Case Western Reserve University and University Hospitals of Cleveland. Cleveland, OH 44106, U.S.A. (Received 18 August 1994; in revised form 10 October 1994)
Two HC-labeled radiopharmaceuticals which are routinely used in clinical PET procedures are [llC]acetate and [tlC]methionine. In implementing published procedures for their synthesis, modifications of the procedures and chemistry were made to create a more rapid and reproducible production process for routine clinical application We report simplified methods which allow a single apparatus to be used for all llC syntheses which use methyl iodide, Grignard reagents, or acetone as labeled synthetic precursors. A reliable TLC quality control method for [llC]acetat e is also reported.
A Scanditronix MC-17 cyclotron equipped with the manufacturer's standard HC target was used. The 14N(p,~)IIc reaction with 17MeV protons was used to produce labeled carbon dioxide. Zero grade (analyzed) nitrogen gas (Gas Technics) was used as the target gas, and the target was periodically conditioned by irradiation of 2% zero grade oxygen in zero grade nitrogen. L-Homocysteine thiolactone hydrochloride was obtained from Sigma Chemical Co., and other materials were obtained from Aldrich Chemical Co. and Fisher Scientific and used without further purification except as noted. HPLC was performed using columns and conditions noted below with a Spectra-Physics SP8700 or Beckman ll0B pump system, and a 2.5 x 7 cm NaI radiation detector (Harshaw) with associated electronics and rate meter (Canberra), with (as appropriate) a Knauer Differential Refractometer (Type 731.980) or a Hewlett-Packard HP1050 multi-wavelength detector, or a Dionex pulsed electrochemical detector. Acetate
Introduction Available syntheses of [llC]acetate (Pike et al.. 1981a,b, 1984; Del Fiore et al., 1984) from a corresponding Grignard reagent rely on purification of the product by organic extraction. This requires aqueous and organic phases to be separated remotely in a shielded enclosure. Although the method has been successfully applied in several laboratories, the extraction was difficult in our existing synthetic apparatus. Even with specialized apparatus, considerable operator skill and precision was required, so the procedure was prone to error. For reliable routine production of a variety of radiopharmaceuticals in limited laboratory space it is desirable to have all ~C labeling procedures take place in a single synthetic apparatus, so a simple purification sequence which would be easily adaptable to the apparatus and to automation was desired. Further, quality control by HPLC was accurate but inconvenient. Available TLC methods did not reliably distinguish acetate from carbonate, especially when low levels of carbonate contamination were present. A reliable TLC method for quality control was needed. Methionine was another radiopharmaceutical of interest for routine use. Two procedures for the synthesis of [lIC]methionine have been reported. Comar et al. (1976), Berger et aL (1979, 1980) and Meyer et al. (1982, 1983) methylated the precursors L-homocysteine thiolactone or DL-homocysteine in the presence of proportionally large amounts of NaOH (60:1 mole ratio) to produce labeled methionine. Langstrom and Davis (Langstrom and Lundquist, 1976; Langstrom et aL, 1987; Davis et al., 1982) converted S-benzyl cysteine to the sulfide using sodium in liquid ammonia before methylation. All approaches used high pressure liquid chromatography for purification of the product. Though all of these approaches provide satisfactory results to their users, we have made minor modifications which significantly altered the routine procedure. We have adapted the simpler method of Comar to allow a simplified purification method which is faster and does not
A Grignard reagent solution (0.05 M methyl magnesium bromide in ether) was prepared by adding 0.05 g clean Mg to 100 mL ether distilled under Ar from calcium hydride. Bromomethane was bubbled into the mixture slowly for 30 min. The reaction mixture was gently heated and stirred for 3 h under At, then collected through Teflon tubing into small Ar-flushed bottles. The reagent solution could be stored at 5°C for several months. Tetrahydrofuran (THF) was freshly distilled under Ar from sodium and benzophenone before use. HPLC was performed on an Alltech 250 x 4.6 mm Adsorbosphere SAX column. The elution solvent was 0.005 M NaH2PO4 at a flow rate of 2 mL/min (R.T.: [llC]acetate, 8 rain; carbonate 3 rain). Bromide was measured on an Alltech Universal Anion column eluted with 2.5mM aqueous 2-hydroxybenzoic acid adjusted to pH 7.9 with sodium hydroxide (R.T.: 6 rain) and magnesium was measured on an Alltech Universal Cation column eluted with 3mM aqueous methanesulfonic acid (R.T.: 9 rain). Gas chromatography was used to analyze the product solvent. It was performed on a Hewlett-Packard 5890 instrument using a thermal conductivity detector and a Porapak N (5m x 3ram) column, eluted with 15 mL/min He at 175°C. Basic thin layer chromatography (TLC) silica plates were prepared by thoroughly wetting standard plates (Merck 0.25mm, 5 x 10 cm silica) by brief immersion in a saturated solution of NaOH in methanol and then drying them in air. They could then be stored indefinitely. NaOH was added to test solutions (to bring them to 0.1 N NaOH) before their application to the basic TLC plates and elution with methanol (Rf: acetate, 0.8; carbonate, 0). Figure 1 shows the schematic diagram of the apparatus used for all ltC syntheses. The 11CO2 was trapped under vacuum in a coil cooled in liquid nitrogen and then transferred to the reaction vessel with a stream of He. It passed through a trap (Tewson et aL, 1989) to remove oxides of nitrogen and was dried by a trap containing P205. It was collected in a solution of 50/t L of 50 mM Grignard solution added to 200 pL of freshly distilled dry THF. The reaction was immediately quenched by adding 100 #L ethanol and 173
Technical Note
174
the solution was evaporated to dryness at 100°C. Additional (2 x 200/~L) portions of ethanol were added and evaporated. Water (2 x 1 mL) was added to rinse the vessel, and the mixture was passed through a pre-wet silica cartridge into a second vessel. The solution was acidified with 150 # L of 1 M HC1 and He gas was bubbled through it for 1 rain, rapidly removing [llC]carbonate. The acid was neutralized with 200#L 1M NaHCO 3 (pH7.8) solution and the product filtered through a 0.22 #m sterile, sterilizing filter. Quality control was performed by adding several drops of product solution to 0.5 mL of 0.1 N NaOH which was then spotted on a basic TLC plate and eluted (10min) as described above. Methionine
HPLC for product analysis was performed using the system described above with an Alltech analytical reverse phase Econosil C-18 column (250 x 4.6mm), eluted with 0.001 M NaH2PO 4 adjusted to pH 3.0 with phosphoric acid (RT: methionine, 3 min; methanol, 2 min). The product was detected by radioactivity and by U.V. absorbance of added standard methionine at 220 rim. For routine quality control, the product pH was verified (7 < pH < 8.5) with indicator (1-10 pH) paper and purity was measured by TLC on Merck 0.25 mm, 5 × 10 cm silica plates eluted with 2% NH4OH in methanol. The radioactive product was measured on a Berthold linear TLC analyzer. Methionine carrier was added to the product or chromatographed separately and was detected with iodine vapor. The same apparatus was used for the [HC]acetate synthesis (Fig. 1)0 but sodium carbonate was substituted for silica in the trap in order to remove HI vapor. Labeled carbon dioxide was prepared as above and collected in a solution of 2 #mol LiA1H4 in 200 #L freshly distilled THF. After evaporating the THF and cooling the vessel, 400 #L of 57% HI was added (Langstrom et al., 1976). Labeled methyl iodide was distilled at 110°C into the second reaction vessel aided by a He gas flow (20 mL/min). It was collected at - 7 0 ° C in a solution of 6.5/~mol L-homocysteine thiolactone .HC1 in 100 #1 dry THF, mixed with a solution of 5 # L of 5 M aqueous NaOH (25 #mol) in 50 # L of ethanol. The mixture was refluxed for 10min at 75°C, then the temperature was increased to 150°C to evaporate the solution to dryness under He gas flow. Ethanol (2 x 200 ~tL) was added and evaporated to remove volatile materials ([llC]iodomethane, [lIC]methanol and THF). The residue
ROW
was taken up in 100 #L of 1 M HC1, neutralized with 300 #L 1 M NaHCO3 and filtered through a sterile 0.2 #m filter into a sterile syringe or vial. Two 2.0 mL portions of sterile saline (USP) were added and used to rinse residual product into the collection vessel.
Results and Discussion The change in the acetate synthesis compared to previous methods was that solvent and volatile impurities were removed by evaporation with ethanol after the Grignard reaction and before acidification. This method eliminated the need for liquid extraction, and allowed complete removal of radiochemical impurities and solvents. One evaporation of 200 #L ethanol reduced the THF content of the product solution below 0.5%, and a second evaporation left no detectable THF in the product. No labeled acetate (CH3 ltCOO - + MgBr) was lost during the procudure. Most of the salts created as reaction byproducts were removed after acidification by filtration through a bed of silica. Traces of Br- and Mg +÷ remained, but in amounts (2-20 #g) that are negligible from the standpoint of toxicity or pharmacological effects (USDA recommended daily allowance for Mg is 300 mg). The use of separate columns to specifically absorb Mg and bromide was reported by Meyer et al. (1993) in a conceptually similar system. In addition to these additional columns, that system also employed phosphoric acid, a longer heating cycle for CO2 removal, and HPLC for quality control. [llC]Carbonate was easily removed after acidification of the mixture, without significant loss of labeled acetate. The purification procedure was therefore effective, and easily manageable by a remotely controlled or automated system. HPLC systems as previously described by others can be used if desired to remove the traces of salt remaining in the product, but are not necessary to achieve a satisfactory injectable material. A simple thin layer chromatography (TLC) procedure was developed to detect any [HC]-carbonate, the only potential radiochemical impurity. Measurement of trace carbonate impurity was initially difficult due to its volatility under acid conditions and to the similarity of its chromatographic properties with those of acetate. This problem was solved by using strongly basic silica TLC plates. Tests of the procedure using standardized mixtures of labeled carbonate and acetate showed that the method was accurate, and that the limit of detectability was well under 0.1%. With the standard
terrors
Met~ CarlTIdge
~-~
Fig. 1. Synthetic apparatus used to prepare all HC-labeled products. The HPLC apparatus shown is optional for this work, though it is used in other preparations.
Technical Note
9O, 80 70.
o~,
60.
~" 4o
10 0
, 2
.
, 4
.
t
.
6
, 8
'
,
•
10
, 12
Mote Ratio: N a O H / H o m o ~ s t e i n e Thiolactone
Fig. 2. Yield of methionine as a function of the mole ratio of sodium hydroxide to homocysteine thiolactone. procedure, the amount of labeled carbonate present was never above 2%, with a normal range of 0.04-1.2%. The procedure gave sterile, pyrogen-free, isotonic and radiochemically pure [HC]-acetate in 60% chemical yield (based on I1CO2) within 15min from the end of bombardment. Typically, an irradiation of 30 #A for 30rain produced 1700 mCi of HC at the end of bombardment and 600 mCi of acetate and end of synthesis. The significant improvement in the methionine process was the marked reduction in the quantity of sodium hydroxide in the reaction mix compared to published methods. The reduction was achieved by reducing the quantity of homocysteine thiolactone as well as the NaOH stoichiometry (4: 1 mole ratio NaOH:thiolactone). This increased the yield of methionine with respect to methyl iodide and, more importantly, it allowed us to simplify the purification process. Figure 2 shows the relationship of the NaOH/thiolactone ratio to the chemical yield of the reaction. Reduced Na content allowed us to produce an isotonic solution without an HPLC purification, and this saved valuable time and simplified the synthetic apparatus while maintaining the final radiochemical purity. In this procedure, all radioactive and undesirable organic impurities were removed by evaporation. A trace of the synthetic precursor, L-homocysteine thiolactone, which could have been removed by HPLC, was allowed to remain in the product. L-homocysteine thio. lactone is a normal metabolite which is present in human serum (1.67#mol/mL; McKully and Vezeridis, 1988) in a concentration similar to that of the injectable product (1.5#mol/mL). The maximum quantity which could be injected in this product (6.5 ~mol) is negligible in comparison to the quantity normally present in the blood pool, and therefore there is no need to remove it. References
Berger G., Maziere M., Knipper R., Prenant C. and Comar D. (1979) Automated synthesis of HC-labeled 8
175
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