Improving the stability of 11C–labeled L-methionine with ascorbate
Carbon-11 labeled L-methionine (11C–MET) is a popular tracer used in the clinic for imaging brain tumors with positron emission tomography. However, the stability of 11C–MET in its final formulation is not well documented in literature. Recently, we observed fast degradation of HPLC-purified 11C–MET over time, and systematic investigation was conducted to identify the cause.
In this study, we verified the degraded product as 11C–labeled methionine sulfoxide (11C–METSO). To minimize oxidation, ascorbate (100 ppm) was added to the HPLC eluant, and the resulting HPLC-purified 11C–MET was stable in the final formulation solution without noticeable degradation for up to 1 h after the end of synthesis.
Our data suggest that to minimize degradation, ascorbate can be added to the 11C–MET formulation solution especially if it is not administered into patients soon after the end of synthesis.
KeywordsC-11 L-methionine Stability Homocysteine, Methionine sulfoxide Oxidation Ascorbate Positron emission tomography
Carbon-11 labeled methyl iodide
Carbon-11 labeled L-methionine
Carbon-11 labeled methionine sulfoxide
End of synthesis
High performance liquid chromatography
Positron emission tomography
Part per million
Carbon-11 labeled L-methionine (11C–MET) is a promising tracer for imaging brain tumors with positron emission tomography (PET) (Watanabe et al. 2016; Maffione et al. 2009; Glaudemans et al. 2013). Its synthesis has been previously optimized by 11C–methylation of L-homocysteine in solution or on a C-18 Sep-Pak cartridge with or without HPLC purification (Pascali et al. 1999; Tang et al. 2004; Lodia et al. 2007; Lodi et al. 2008; Boschi et al. 2009; Cheung et al. 2009; Quincoces et al. 2010; Pascali et al. 2011; Bogni et al. 2003; Nagren et al. 1998; Oh et al. 1998; Fukumura et al. 2004). While most groups reported > 97% radiochemical purity of 11C–MET at the end of synthesis (EOS) even without HPLC purification, the stability of 11C–MET in the final formulation solution has not been well documented in literature (Pascali et al. 1999; Tang et al. 2004; Lodia et al. 2007; Lodi et al. 2008; Boschi et al. 2009; Cheung et al. 2009; Quincoces et al. 2010; Pascali et al. 2011; Bogni et al. 2003; Nagren et al. 1998; Oh et al. 1998).
In this methodology article, we communicate our experience on the preparation of HPLC-purified 11C–MET. We report here the instability of HPLC-purified 11C–MET, our systematic investigation to find out the cause of rapid degradation, and the strategy to improve the stability of 11C–MET in the final formulation solution.
Results and discussion
To set up 11C–MET production at our institution, we used the simplest method, Sep-Pak cartridge without HPLC purification (Gomzina et al. 2011), for our initial attempt. However, we obtained much lower radiochemical purity (< 90%, data not shown) as compared to > 97% reported by others (Pascali et al. 1999; Tang et al. 2004; Lodia et al. 2007; Lodi et al. 2008; Boschi et al. 2009; Cheung et al. 2009; Quincoces et al. 2010; Pascali et al. 2011; Bogni et al. 2003). For the preparation of 11C–MET using Sep-Pak cartridge without HPLC purification, L-homocysteine and unhydrolyzed L-homocysteine thiolactone end up in the final formulation solution as well. In order to achieve higher chemical and radiochemical purities, we decided to use HPLC purification for subsequent preparations of 11C–MET.
We successfully verified the degradation of HPLC-purified 11C–MET was due to the formation of 11C–METSO. Presence of L-homocysteine or EtOH in the final 11C–MET formulation solution could slow down the degradation of 11C–MET. Adding ascorbate to the HPLC solvent greatly improved the radiochemical purity and stability of HPLC-purified 11C–MET solution. This could be very useful especially if 11C–MET is not used immediately after EOS. The tested concentration (100 ppm) contains only ~1.4 mg of ascorbate in the entire dose (~13.5 mL). It is safe for administration as this mass of ascorbate is much lower than the usual therapeutic parenteral dose (100–250 mg).
METSO and sodium phosphate monobasic were purchased from Sigma-Aldrich (Oakville, Canada). For preparation of the QC HPLC solvent and the phosphate buffer used to elute the reaction mixture off the cartridge, sodium phosphate monobasic was diluted with water to the specified concentrations without adjusting pH. Vitamin C injection (ascorbic acid, 250 mg/mL) and sodium phosphate injection (3 mmol/mL) were purchased from Sandoz (Boucherville, Canada). Saline injection (0.9% NaCl) was purchased from Baxter (Mississauga, Canada). The semi-preparative HPLC solvents were prepared by mixing sodium phosphate injection and saline injection (with or without vitamin C injection) to the specified concentrations. All other chemicals and solvents were obtained from commercial sources, and used without further purification. Sep-Pak tC18 Plus Short cartridges (400 mg) were obtained from Waters (Milford, MA). C-11 methane was produced by 18-MeV proton bombardment of an N2/H2 (10% H2 in N2) target using an Advanced Cyclotron Systems Inc. (Richmond, Canada) TR19 cyclotron. C-11 methane was converted to C-11 methyl iodide (11CH3I) in gas phase using a GE (Chicago, IL) TRACER FX C Pro module. Purification of 11C–MET was conducted using the HPLC component of the synthesis module on a Phenomenex (Torrance, CA) Aqua C18 semi-preparative column (5 μ, 250 × 10 mm). The HPLC solvent was phosphate-buffered saline (PBS, 3.93 mM) or PBS containing 100 ppm of ascorbate, and the flow rate was 3.0 mL/min. Millex-GS 0.22 μm sterile filter was purchased from EMD Millipore (Billerica, MA). Radioactivity was measured using a Capintec (Ramsey, NJ) CRC®-Ultra R dose calibrator. Mass analysis was performed using an AB SCIEX (Framingham, MA, USA) 4000 QTRAP mass spectrometer system with an ESI ion source.
Synthesis and purification of 11C–MET
The tC18 Sep-Pak cartridge was preconditioned with EtOH (5 mL) and sterile water (10 mL). The remaining water in the cartridge was pushed out with air (10 mL). Five minutes before EOS, 85 μL of L-homocysteine thiolactone hydrochloride aqueous solution (25 mg in 600 μL water) was mixed with 200 μL of NaOH solution (0.7 mL of 10 N NaOH aqueous solution diluted with 4.3 mL water and 5.0 mL EtOH). From this, 200 μL of the mixed solution was loaded to the tC18 Sep-Pak cartridge. After passing 11CH3I by helium (15 mL/min) through the tC18 Sep-Pak cartridge, the reaction mixture was eluted off the cartridge with phosphate buffer (50 mM, 2 mL) and purified by HPLC. The eluate fraction (~ 1.5 mL) containing 11C–MET was collected, diluted with HPLC eluant (12 mL), and passed through a Millex-GS sterile filter into a final product vial.
Quality control of 11C–MET
Chemical purity, radiochemical purity and radiochemical identity of 11C–MET and by-products were determined using an Agilent (Santa Clara, CA) HPLC system equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 220 nm), and a Bioscan (Washington, DC) NaI scintillation detector. The operation of the Agilent HPLC system was controlled using the Agilent ChemStation software. The HPLC column used was a Phenomenex Luna C18 analytical column (5 μ, 250 × 4.6 mm). The HPLC solvent was phosphate buffer (1 mM, pH 3), and the flow rate was 1.0 mL/min.
This work was supported by the Canadian Institutes of Health Research (FDN-148465) and the Leading Edge Endowment Fund.
MW, DW, FB and KSL conceived and designed the complete study. MW, LL, KF, JGG, ZZ, CZ and WE conducted the experiments. MW, LL, ZZ, CZ and KSL summarized and interpreted the data. The manuscript was drafted by KSL with critical revisions from MW, LL, KF, JGG, ZZ, CZ, and WE. All authors read and approved the final manuscript.
Consent for publication
The authors declare that they have no competing interests.
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