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Improving the stability of 11C–labeled L-methionine with ascorbate Woods, Michael; Leung, Leo; Frantzen, Kari; Garrick, Jennifer G; Zhang, Zhengxing; Zhang, Chengcheng; English, Wade; Wilson, Don; Bénard, François; Lin, Kuo-Shyan Oct 4, 2017

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METHODOLOGY Open AccessImproving the stability of 11C–labeled L-methionine with ascorbateMichael Woods1, Leo Leung1, Kari Frantzen1, Jennifer G. Garrick1, Zhengxing Zhang2, Chengcheng Zhang2,Wade English1, Don Wilson1, François Bénard1,2,3 and Kuo-Shyan Lin1,2,3** Correspondence: klin@bccrc.ca1Department of Functional Imaging,BC Cancer Agency, Vancouver, BC,Canada2Department of MolecularOncology, BC Cancer Agency,Vancouver, BC, CanadaFull list of author information isavailable at the end of the articleAbstractBackground: Carbon-11 labeled L-methionine (11C–MET) is a popular tracer used inthe 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, andsystematic investigation was conducted to identify the cause.Results: In this study, we verified the degraded product as 11C–labeled methioninesulfoxide (11C–METSO). To minimize oxidation, ascorbate (100 ppm) was added to theHPLC eluant, and the resulting HPLC-purified 11C–MET was stable in the final formulationsolution without noticeable degradation for up to 1 h after the end of synthesis.Conclusions: Our data suggest that to minimize degradation, ascorbate can be addedto the 11C–MET formulation solution especially if it is not administered into patientssoon after the end of synthesis.Keywords: C-11 L-methionine, Stability, Homocysteine, Methionine sulfoxide, Oxidation,Ascorbate, Positron emission tomographyBackgroundCarbon-11 labeled L-methionine (11C–MET) is a promising tracer for imaging braintumors with positron emission tomography (PET) (Watanabe et al. 2016; Maffione etal. 2009; Glaudemans et al. 2013). Its synthesis has been previously optimized by11C–methylation of L-homocysteine in solution or on a C-18 Sep-Pak cartridge withor 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; Pascaliet 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 theend of synthesis (EOS) even without HPLC purification, the stability of 11C–MET inthe final formulation solution has not been well documented in literature (Pascali etal. 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).Bogni et al. reported the formation of a minor amount (< 6%) of C-11 L-methioninesulfoxide (11C–METSO) at 1 h after EOS in 11C–MET solution prepared either with orwithout HPLC purification (Fig. 1) (Bogni et al. 2003). The degradation of 11C–METEJNMMI Radiopharmacy                and Chemistry © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, andindicate if changes were made.Woods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 DOI 10.1186/s41181-017-0032-xwas attributed to radiolysis as the degradation rate was affected by total radioactivity andchemical composition (ethanol and L-homocysteine) of the 11C–MET solutions.Fukumura et al. observed fast degradation of HPLC-purified 11C–MET solution withradiochemical purity at 91.2 and 72.9% for samples assayed at EOS and 20 min after EOS,respectively (Fig. 1b) (Fukumura et al. 2004). The instability of 11C–MET was likelycaused by radiolysis as samples with higher specific activity also showed significantly fasterdegradation rates. The degraded radioactive product was not identified in this report.However, it was shown that adding ethanol (EtOH, 1.5%) and Tween 80 (3%) into the finalformulation solution (in saline) or using saline containing ascorbate (1000 ppm) as theeluant improved the radiochemical purity of HPLC-purified 11C–MET to 99.9%, and nosignificant degradation was observed over 1 h after EOS (Fukumura et al. 2004).In this methodology article, we communicate our experience on the preparation ofHPLC-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 strategyto improve the stability of 11C–MET in the final formulation solution.Results and discussionTo set up 11C–MET production at our institution, we used the simplest method, Sep-Pakcartridge without HPLC purification (Gomzina et al. 2011), for our initial attempt.However, we obtained much lower radiochemical purity (< 90%, data not shown) as com-pared 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 withoutHPLC purification, L-homocysteine and unhydrolyzed L-homocysteine thiolactone endup in the final formulation solution as well. In order to achieve higher chemical and radio-chemical purities, we decided to use HPLC purification for subsequent preparations of11C–MET.Fig. 1 Reported radiochemical purity at the end of synthesis and stability of 11C–MET prepared withouta or with b HPLC purificationWoods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 2 of 9Using phosphate-buffered saline (PBS, 3.93 mM, 3.0 mL/min) as the HPLC eluantand a C18 column (Luna C18 semi-preparative column, 5 μ, 250 × 10 mm) 11C–METwas obtained in 19 ± 4% (n = 4) decay-corrected radiochemical yield from 11CH3I with93.5 ± 0.6% (n = 4) radiochemical purity and 21.0 ± 5.1 GBq/μmol specific activity atthe end of synthesis (EOS). The analytical HPLC column was a Luna C18 column(5 μ, 250 × 4.6 mm), the eluant was phosphate buffer (1 mM, pH 3), and the flow ratewas 1.0 mL/min. A minor radioactive by-product was observed at tR = ~3.3 min(Fig. 2a). No residual L-homocysteine or L-homocysteine thiolactone was detected inthe final 11C–MET solution as monitored by UV detector (set at 220 nm). The radio-chemical purity of 11C–MET quickly dropped to 75 ± 6% (n = 4) at 1 h after EOS withconcurrent increase of the radioactive by-product (Fig. 2b). Our radiochemical puritydata (93.5% at EOS and 75% at 1 h after EOS) were lower than the data (> 99% atEOS and > 96% at 1 h after EOS) reported by Bogni et al. (Bogni et al. 2003), but werecomparable to the data (91.2% at EOS and 72.9% at 20 min after EOS) reported byFukumura et al. (Fukumura et al. 2004).To our knowledge, there was only one report on degradation of 11C–MET preparedusing Sep-Pak cartridge without HPLC purification (Bogni et al. 2003). A minoramount (< 4%) of 11C–METSO was formed over a 1-h period after EOS (Bogni et al.2003). Presumably, the remaining L-homocysteine ending up in the final formulationsolution could serve as a free radical scavenger, and prevent degradation of 11C–MET.To verify this hypothesis, we added 0.5 mg of L-homocysteine in the product collectionFig. 2 Representative radio-HPLC chromatograms of 11C–MET (tR = ~5.6 min) purified by HPLC using PBS asthe eluant: a assayed at EOS and b assayed at 1 h after EOSWoods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 3 of 9vial of the synthesis module. After mixing with the HPLC-purified 11C–MET eluate frac-tion, the mixed solution was passed through a sterile filter and checked by HPLC. Asshown in Fig. 3a, the radiochemical purity of 11C–MET increased to 97.5 ± 1.5% (n = 2) atEOS with a minor radioactive by-product eluted at the same retention (tR = ~3.3 min) ofthe degraded product as shown in Fig. 2b. However, the content of this radioactive by-product still slowly increased over time even in the presence of L-homocysteine(radiochemical purity of 11C–MET: 91.1 ± 8.3% at 1 h after EOS, Fig. 3b).Next, we tried to identify the radioactive degradation product in our HPLC-purified11C–MET solution. Previously, 11C–METSO was reported to be the degradation prod-uct of 11C–MET prepared with or without HPLC purification (Bogni et al. 2003). Toverify this, we co-injected the degraded 11C–MET solution (1 h after EOS) with METand METSO into HPLC. As shown in Fig. 4, the UV peak of METSO co-eluted withthe radio peak of the radioactive by-product (tR = ~3.3 min), confirming11C–METSOwas indeed the degradation by-product of our HPLC-purified 11C–MET. In addition,we also conducted mass analysis for the decayed product solution, and confirmed thepresence of METSO (see Additional file 1: Figure S1).Previously, Bogni et al. suggested that 11C–MET prepared without HPLC purificationcould be stabilized by the remaining EtOH and L-homocysteine in the final product solu-tion (Bogni et al. 2003). Fukumura et al. also demonstrated that HPLC-purified 11C–METcould be stabilized by the addition of EtOH (1.5%) and Tween 80 (3%) to the final productsolution (Fukumura et al. 2004). Since EtOH is relatively nontoxic and readily available,we tested if EtOH alone could be used to stabilize the HPLC-purified 11C–MET solution.Fig. 3 Representative Radio-HPLC chromatograms of 11C–MET purified by HPLC using PBS as the eluantand spiked with homocysteine: a assayed at EOS and b assayed at 1 h after EOSWoods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 4 of 9We added EtOH (4%) immediately after the 11C–MET-containing HPLC eluatefraction was collected in the final product vial, and checked radiochemical purity of11C–MET over time. The radiochemical purities of EtOH-containing 11C–MET solu-tion were 94.7 ± 3.3% and 77.7 ± 16.1% (n = 2) at EOS and 1 h after EOS, respectively.Representative HPLC chromatograms are shown in Fig. 5. These data suggest thatEtOH alone is not very effective to prevent degradation of HPLC-purified 11C–METsolution. The previous findings by others might be due to the combination of EtOHwith L-homocysteine or Tween 80 (Bogni et al. 2003; Fukumura et al. 2004).Interestingly, 11C–METSO was observed in Figs. 2, 3 and 5 even at EOS, suggesting11C–METSO was formed quickly after 11C–MET was separated from L-homocysteineduring HPLC purification. To stabilize 11C–MET final formulation solution andminimize the formation of 11C–METSO even during the HPLC purification process,we added ascorbate directly into HPLC solvent. Instead of 1000 ppm tested byFukumura et al. (Fukumura et al. 2004), we used only 100 ppm of ascorbate. The eluatefraction containing 11C–MET was collected and passed through a sterile filter. 11C–METwas obtained in 22 ± 3% (n = 8) decay-corrected radiochemical yield from 11CH3I with a99.2 ± 0.9% (n = 8) radiochemical purity at EOS. No significant degradation of 11C–METsolution was observed as the radiochemical purity was 98.2 ± 1.7% (n = 8) at 1 h afterEOS (Fig. 6). These data are consistent with the observation of Fukumura et al. (Fuku-mura et al. 2004), and suggest that 100 ppm of ascorbate is sufficient to minimize the for-mation of 11C–METSO.Fig. 4 HPLC chromatograms of co-injecting MET and METSO with 11C–MET solution purified by HPLC usingPBS as the eluant: a UV chromatogram b Radio chromatogramWoods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 5 of 9ConclusionsWe successfully verified the degradation of HPLC-purified 11C–MET was due to theformation of 11C–METSO. Presence of L-homocysteine or EtOH in the final 11C–METformulation solution could slow down the degradation of 11C–MET. Adding ascorbateto 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 usedimmediately after EOS. The tested concentration (100 ppm) contains only ~1.4 mg ofascorbate in the entire dose (~13.5 mL). It is safe for administration as this mass of as-corbate is much lower than the usual therapeutic parenteral dose (100–250 mg).MethodsGeneral methodsMETSO and sodium phosphate monobasic were purchased from Sigma-Aldrich(Oakville, Canada). For preparation of the QC HPLC solvent and the phosphate bufferused to elute the reaction mixture off the cartridge, sodium phosphate monobasicwas diluted with water to the specified concentrations without adjusting pH. VitaminC injection (ascorbic acid, 250 mg/mL) and sodium phosphate injection (3 mmol/mL)were purchased from Sandoz (Boucherville, Canada). Saline injection (0.9% NaCl) waspurchased from Baxter (Mississauga, Canada). The semi-preparative HPLC solventswere prepared by mixing sodium phosphate injection and saline injection (with orFig. 5 Radio-HPLC chromatograms of 11C–MET purified by HPLC using PBS as the eluant and spiked withEtOH (4%): a assayed at EOS and b assayed at 1 h after EOSWoods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 6 of 9without vitamin C injection) to the specified concentrations. All other chemicals andsolvents 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 inN2) target using an Advanced Cyclotron Systems Inc. (Richmond, Canada) TR19 cyclo-tron. C-11 methane was converted to C-11 methyl iodide (11CH3I) in gas phase using aGE (Chicago, IL) TRACER FX C Pro module. Purification of 11C–MET was conductedusing 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 wasphosphate-buffered saline (PBS, 3.93 mM) or PBS containing 100 ppm of ascorbate, andthe flow rate was 3.0 mL/min. Millex-GS 0.22 μm sterile filter was purchased fromEMD Millipore (Billerica, MA). Radioactivity was measured using a Capintec(Ramsey, NJ) CRC®-Ultra R dose calibrator. Mass analysis was performed using an ABSCIEX (Framingham, MA, USA) 4000 QTRAP mass spectrometer system with an ESIion source.Synthesis and purification of 11C–METThe 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). Fiveminutes before EOS, 85 μL of L-homocysteine thiolactone hydrochloride aqueousFig. 6 Representative radio-HPLC chromatograms of 11C–MET purified by HPLC using PBS containing100 ppm of ascorbate as the eluant: a assayed at EOS and b assayed at 1 h after EOSWoods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 7 of 9solution (25 mg in 600 μL water) was mixed with 200 μL of NaOH solution (0.7 mL of10 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 passing11CH3I by helium (15 mL/min) through the tC18 Sep-Pak cartridge, the reaction mixturewas 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 HPLCeluant (12 mL), and passed through a Millex-GS sterile filter into a final product vial.Quality control of 11C–METChemical purity, radiochemical purity and radiochemical identity of 11C–MET and by-products were determined using an Agilent (Santa Clara, CA) HPLC system equippedwith a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at220 nm), and a Bioscan (Washington, DC) NaI scintillation detector. The operation of theAgilent HPLC system was controlled using the Agilent ChemStation software. The HPLCcolumn used was a Phenomenex Luna C18 analytical column (5 μ, 250 × 4.6 mm). TheHPLC solvent was phosphate buffer (1 mM, pH 3), and the flow rate was 1.0 mL/min.Additional fileAdditional file 1: Figure S1. Identification of METSO from decayed 11C–MET solution. (DOCX 260 kb)Abbreviations11CH3I: Carbon-11 labeled methyl iodide;11C–MET: Carbon-11 labeled L-methionine; 11C–METSO: Carbon-11 labeledmethionine sulfoxide; EOS: End of synthesis; EtOH: Ethanol; HPLC: High performance liquid chromatography;NaOH: Sodium hydroxide; PBS: Phosphate-buffered saline; PET: Positron emission tomography; ppm: Part per million;tR: Retention time; UV: Ultraviolet lightAcknowledgmentsThis work was supported by the Canadian Institutes of Health Research (FDN-148465) and the Leading EdgeEndowment Fund.Authors’ contributionsMW, DW, FB and KSL conceived and designed the complete study. MW, LL, KF, JGG, ZZ, CZ and WE conducted theexperiments. MW, LL, ZZ, CZ and KSL summarized and interpreted the data. The manuscript was drafted by KSL withcritical revisions from MW, LL, KF, JGG, ZZ, CZ, and WE. All authors read and approved the final manuscript.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Author details1Department of Functional Imaging, BC Cancer Agency, Vancouver, BC, Canada. 2Department of Molecular Oncology,BC Cancer Agency, Vancouver, BC, Canada. 3Department of Radiology, University of British Columbia, Vancouver, BC,Canada.Received: 30 July 2017 Accepted: 11 September 2017ReferencesBogni A, Bombardieri E, Iwata R, Cadini L, Pascali C. Stability of L-[S-methyl-11C]methionine solutions. J Radioanal NuclChem. 2003;256:199–203.Boschi S, Lodi F, Cicoria G, Ledesma JR, Knopp R, Rizzello A, Di Pierro D, Trespidi S, Marengo M. Development of amodular system for the synthesis of PET [11C]labelled radiopharmaceuticals. Appl Radiat Isot. 2009;67:1869–73.Cheung M-K, Ho C-L. A simple, versatile, low-cost and remotely operated apparatus for [11C]acetate, [11C]choline,[11C]methionine and [11C]PIB synthesis. Appl Radiat Isot. 2009;67:581–9.Woods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 8 of 9Fukumura T, Nakao R, Yamaguchi M, Suzuki K. Stability of 11C-labeled PET radiopharmaceuticals. Appl Radiat Isot. 2004;61:1279–87.Glaudemans AWJM, Enting RH, Heesters MAAM, Dierckx RAJO, van Rheenen RWJ, Walenkamp AME, Slart RHJA. Value of11C-methionine PET in imaging brain tumours and metastases. Eur J Nucl Med Mol Imaging. 2013;40:615–35.Gomzina NA, Kuznetsova OF. L-[Methyl-(11C)]-methionine of high enantiomeric purity production via online-11C-methylation of L-homocysteine thiolactone hydrochloride. Russ J Bioorg Chem. 2011;37:191–7.Lodi F, Rizzello A, Trespidi S, Di Pierro D, Marengo M, Farsad M, Fanti S, Al-Nahhas A, Rubello D, Boschi S. Reliability andreproducibility of N-[11C]methyl-choline and L-(S-methyl-[11C])methionine solid-phase synthesis: a useful andsuitable method in clinical practice. Nucl Med Commun. 2008;29:736–40.Lodi F, Trespidi S, Di Pierro D, Marengo M, Farsad M, Fanti S, Franchi R, Boschi S. A simple Tracerlab modulemodification for automated on-column [11C]methylation and [11C]carboxylation. Appl Radiat Isot. 2007;65:691–5.Maffione AM, Nanni C, Ambrosini V, Trespidi S, Lopci E, Allegri V, Castellucci P, Montini G, Boschi S, Fanti S. 11C-Methionine PET/CT in central nervous system tumours: a review. Curr Radiopharma. 2009;2:160–4.Nagren K, Halldin C. Methylation of amide and thiol functions with [11C]methyl triflate, as exemplified by [11C]NMSP,[11C]flumazenil and [11C]methionine. J Label Compd Radiopharm. 1998;41:831–41.Oh S-J, Choe YS, Kim YS, Choi Y, Kim SE, Lee KH, Ha H-J, Kim B-T. Development of an automated system for the routinepreparation of carbon-11 labeled radiopharmaceuticals. Bull Kor Chem Soc. 1998;19:952–6.Pascali C, Bogni A, Cucchi C, Laera L, Crispu O, Maiocchi G, Crippa F, Bombardieri E. Detection of additional impuritiesin the UV-chromatogram of L-[S-methyl-11C]methionine. J Radioanal Nucl Chem. 2011;288:405–9.Pascali C, Bogni A, Iwata R, Decise D, Crippa F, Bombardieri E. High efficiency preparation of L-[S-methyl-11C]methionineby on-column [11C]methylation on C18 sep-Pak. J Label Compd Radiopharm. 1999;42:715–24.Quincoces G, Lopez-Sanchez L, Sanchez-Martınez M, Rodrıguez-Fraile M, Penuelas I. Design and performance evaluationof single-use whole-sterile “plug & play” kits for routine automated production of [11C]choline and [11C]methioninewith radiopharmaceutical quality. Appl Radiat Isot. 2010;68:2298–301.Tang G-H, Wang M-F, Tang X-L, Luo L, Gan M-Q. Automated synthesis of (S-[11C]-methyl)-L-methionine and (S-[11C]-methyl)-L-cycteine by on-column [11C]methylation. J Nucl Radiochem. 2004;26:77–83.Watanabe A, Muragaki Y, Maruyama T, Shinoda J, Okada Y. Usefulness of 11C-methionine positron emissiontomography for treatment-decision making in cases of non-enhancing glioma-like brain lesions. J Neuro-Oncol.2016;126:577–83.Woods et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:13 Page 9 of 9

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