This study was partially supported by a grant 18-05 from the Alzheimer&#8217;s Society of Canada (for A. K.) and Brain Canada Foundation with support from Health Canada. The authors would like to acknowledge the McGill University Faculty of Medicine, Montreal Neurological Institute and McConnell Brain Imaging Centre for support of this work. We also thank Mrs. Monica Lacatus-Samoila for help with quality control procedures and Drs. Jean-Paul Soucy and Gassan Massarweh for access to radioisotopes and the radiochemistry facility.

1. Preparation of buffers and eluents
<ol>
	<li>Dissolve 2.72 g of sodium acetate trihydrate in 100 mL of water to prepare 0.2 M sodium acetate solution (solution A).</li>
	<li>Dissolve 11.4 mL of glacial acetic acid in 1 L of water to prepare 0.2 M acetic acid solution (solution B).</li>
	<li>Combine 50 mL of solution A with 450 mL of solution B to prepare the acetate buffer at pH 3.7 (buffer 1) according to the buffer reference center15. Verify the pH of the buffer with pH strips or a pH meter.</li>
	<li>Combine 12.5 mL of absolute ethanol with 87.5 mL of buffer 1 to make 12.5% aqueous EtOH solution (wash 1) in a 100 mL bottle.</li>
	<li>Combine 15 mL of absolute ethanol with 85 mL of buffer 1 to make 15% aqueous EtOH solution (wash 2) in a 100 mL bottle.</li>
	<li>Combine 5 mL of absolute ethanol with 5 mL of buffer 1 to make 50% aqueous EtOH solution (final eluent) and draw 2.5 mL of this solution into a 10 mL syringe.</li>
</ol>
2. Application of the precursor to the cartridge
<ol>
	<li>Pass 10 mL of water followed by 5 mL of acetone through the tC18 cartridge to precondition it.</li>
	<li>Dry the cartridge with a stream of nitrogen at 50 mL/min for 1 min.</li>
	<li>Dissolve 2 mg of the precursor 6-OH-BTA-0 in 1 mL of anhydrous acetone.</li>
	<li>Holding a Luer-tip 250 &#181;L precision glass syringe downwards, withdraw 100 &#181;L of the precursor solution and 50 &#181;L of air cushion on top of the liquid. Remove the needle and apply the precursor solution on the tC18 cartridge from the female end by slowly pushing the plunger all the way down. Do not push the solution any further!</li>
</ol>
3. Setting up the manifold for automated synthesis
<ol>
	<li>Secure the standard 5-port disposable manifold on the synthesis module and assemble it according to the Figure 2 and steps 3.2 - 3.5 below. 
	NOTE: We recommend using acetone-resistant manifolds (see Table of Materials).</li>
	<li>Port 1 has two positions. Connect the horizontal inlet to the automated dispenser fitted with a 20 mL syringe. Connect the vertical inlet to the bottle with wash 1.</li>
	<li>Connect the output of the module which produces [11C]CH3OTf to port 2 of the manifold.</li>
	<li>Install the tC18 cartridge loaded with precursor 6-OH-BTA-0 between ports 3 and 4.</li>
	<li>Port 5 has two positions. Connect the horizontal outlet to the waste bottle which must hold at least 200 mL. Connect the vertical outlet to the sterile vial for tracer collection via the sterile filter.</li>
</ol>
4. Radiosynthesis of [11C]PiB
CAUTION: All manipulations involving radioactive isotopes must be performed in a lead-shielded hot cell by personnel with adequate training to work with radioactive materials. 
NOTE: This protocol does not cover the details of production of [11C]CO2 in the cyclotron and its conversion into [11C]CH3OTf using the radiochemistry module. These procedures will depend on the individual equipment of the radiochemistry lab and are outside the scope of this protocol. Our PET centre is equipped with an IBA cyclotron, which produces carbon-11 in the chemical form of [11C]CO2 via the 14N(p,&#945;)11C nuclear reaction with a N2/O2 gas mixture (99.5:0.5) in the gas target, and a commercially available module for production of [11C]CH3I via the &#34;dry method&#34; (catalytic reduction to [11C]CH4 followed by radical iodination). [11C]CH3OTf is produced by passing [11C]CH3I over a silver triflate column heated to 175 &#176;C at 20 mL/min.
<ol>
	<li>Deliver [11C]CH3OTf into the manifold through port 2 and pass it through the loaded tC18 cartridge at 20 mL/min output flow regulated by the [11C]CH3OTf module, via ports 3 and 4 and into the waste bottle as shown on Figure 2A.</li>
	<li>Once all the radioactivity has been transferred and trapped on the tC18 cartridge as monitored by the radioactivity detector behind the cartridge holder, stop the flow of gas by closing port 2. Let the cartridge sit for 2 min to complete the reaction.</li>
	<li>Withdraw 19 mL of wash 1 solution (see step 1.4) from the 100 mL bottle into the dispenser syringe through port 1 at 100 mL/min as shown on Figure 2B.</li>
	<li>Dispense 18.5 mL of wash 1 solution from the dispenser through the tC18 cartridge via ports 3 and 4 and into the waste bottle at 50 mL/min as shown on Figure 2C. Ensure the absence of air bubbles in the manifold as they might diminish the separation efficiency.</li>
	<li>Repeat steps 4.3 and 4.4 four times, withdrawing and dispensing 18.5 mL of wash 1 solution each time. The total volume of wash 1 solution passed through tC18 is 92.5 mL; however, it can vary within the 90 - 100 mL range depending on the particular synthesis module used.</li>
	<li>Switch the input line on port 1 from wash 1 to wash 2 solution (see step 1.5).</li>
	<li>Repeat steps 4.3 and 4.4 three times, withdrawing and dispensing 18.5 mL of wash 2 solution each time. The total volume of wash 2 solution passed through tC18 is 55.5 mL. However, it can vary within the 50 - 60 mL range depending on the particular synthesis module used.</li>
	<li>Toggle valve 5 towards the final vial as shown on Figure 2D. Disconnect the line from the dispenser and connect it to the 10 mL syringe containing 2.5 mL of the final eluent solution (50% aqueous EtOH, see step 1.6) and 7.5 mL of air.</li>
	<li>Holding the syringe downwards, manually push the final eluent solution (2.5 mL) followed by air (7.5 mL) through the tC18 cartridge via ports 3 and 4 and into the sterile vial for tracer collection via the sterile filter as shown on Figure 2D.</li>
	<li>Disconnect the empty syringe, connect the 10 mL syringe containing 10 mL of the sterile phosphate buffer (recipe not included as it may vary) and push the entire volume through the tC18 cartridge into the sterile vial as described above (Figure 2D). Disconnect the syringe and flush the line with 10 mL of air using the same syringe.</li>
	<li>Withdraw 0.7 mL of the final tracer formulation and collect samples for quality control procedures (0.1 mL), bacterial endotoxin test (0.1 mL) and sterility (0.5 mL).</li>
</ol>
5. Quality control procedures
CAUTION: Each batch of the radiotracer must be subjected to the appropriate quality control procedures (QC) prior to release to the PET imaging site for administration into human or animal subjects. The authors of this manuscript are not responsible for the compliance of the radiotracer produced at other centers with local health authority regulations.
<ol>
	<li>Perform pre-release QC procedures, which must include tests for radiochemical identity (RCI), radiochemical purity (RCP), chemical purity and molar activity of the tracer as well as residual solvent content and pH of the formulation.</li>
	<li>Determine the RCI, RCP, chemical purity and molar activity by means of analytical HPLC system equipped with UV (monitoring at 350 nm) and radioactivity detectors, and a reversed-phase column. Determine the retention times of 6-OH-BTA-0 and 6-OH-BTA-1 and calibrate the instrument to quantify the content of each compound.</li>
	<li>Determine the residual solvent content by means of analytical gas chromatography system equipped with a capillary column. Determine the retention times of acetone and ethanol and calibrate the instrument to quantify the content of each solvent.</li>
	<li>Perform the bacterial endotoxins test using a cartridge reader equipped with suitable cartridges.</li>
	<li>Perform the sterility analysis of the sample at least 14 day after the synthesis to ensure the absence of bacterial growth or send the sterility sample to a laboratory accredited by the local health authority.</li>
</ol>

<ol>
	<li>Langstrom, B., Lundqvist, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+of+11C-methyl+iodide+and+its+use+in+the+synthesis+of+11C-methyl-L-methionine.">The preparation of 11C-methyl iodide and its use in the synthesis of 11C-methyl-L-methionine.</a> The International journal of applied radiation and isotopes. 27 (7), 357-363 (1976).</li><li>Larsen, P., Ulin, J., Dahlstr&#248;m, K., Jensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+[11C]iodomethane+by+iodination+of+[11C]methane.">Synthesis of [11C]iodomethane by iodination of [11C]methane.</a> Applied radiation and isotopes. 48 (2), 153-157 (1997).</li><li>Jewett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+synthesis+of+[11C]methyl+triflate.">A simple synthesis of [11C]methyl triflate.</a> International journal of radiation applications and instrumentation. Part A, Applied radiation and isotopes. 43 (11), 1383-1385 (1992).</li><li>Wilson, A. A., Garcia, A., Houle, S., Vasdev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+commercial+radiosynthetic+modules+in+captive+solvent+[11C]-methylation+reactions.">Utility of commercial radiosynthetic modules in captive solvent [11C]-methylation reactions.</a> Journal of Labelled Compounds and Radiopharmaceuticals. 52 (11), 490-492 (2009).</li><li>Wilson, A. A., Garcia, A., Jin, L., Houle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotracer+synthesis+from+[(11)C]-iodomethane:+a+remarkably+simple+captive+solvent+method.">Radiotracer synthesis from [(11)C]-iodomethane: a remarkably simple captive solvent method.</a> Nuclear medicine and biology. 27 (6), 529-532 (2000).</li><li>Fedorova, O. S., Vaitekhovich, F. P., Krasikova, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Synthesis+of+[18F]Fluoromethylcholine+for+Positron-Emission+Tomography+Imaging.">Automated Synthesis of [18F]Fluoromethylcholine for Positron-Emission Tomography Imaging.</a> Pharmaceutical Chemistry Journal. 52 (8), 730-734 (2018).</li><li>Jewett, D. M., Ehrenkaufer, R. L., Ram, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+captive+solvent+method+for+rapid+radiosynthesis:+application+to+the+synthesis+of+[1-(11)C]palmitic+acid.">A captive solvent method for rapid radiosynthesis: application to the synthesis of [1-(11)C]palmitic acid.</a> The International journal of applied radiation and isotopes. 36 (8), 672-674 (1985).</li><li>Watkins, G. L., Jewett, D. M., Mulholland, G. K., Kilbourn, M. R., Toorongian, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+captive+solvent+method+for+rapid+N-[11C]methylation+of+secondary+amides:+application+to+the+benzodiazepine,+4'-chlorodiazepam+(RO5-4864).">A captive solvent method for rapid N-[11C]methylation of secondary amides: application to the benzodiazepine, 4'-chlorodiazepam (RO5-4864).</a> International journal of radiation applications and instrumentation. Part A, Applied radiation and isotopes. 39 (5), 441-444 (1988).</li><li>Hockley, B. G., Henderson, B., Shao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27,+Synthesis+of+{11C]Raclopride.">Chapter 27, Synthesis of {11C]Raclopride.</a> Radiochemical Syntheses. , 167-175 (2012).</li><li>Lodi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+reproducibility+of+N-[11C]methyl-choline+and+L-(S-methyl-[11C])methionine+solid-phase+synthesis:+a+useful+and+suitable+method+in+clinical+practice.">Reliability and reproducibility of N-[11C]methyl-choline and L-(S-methyl-[11C])methionine solid-phase synthesis: a useful and suitable method in clinical practice.</a> Nuclear Medicine Communications. 29 (8), 736-740 (2008).</li><li>Jolly, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+"[(11)C]kits"+for+a+fast,+efficient+and+reliable+production+of+carbon-11+labeled+radiopharmaceuticals+for+Positron+Emission+Tomography.">Development of "[(11)C]kits" for a fast, efficient and reliable production of carbon-11 labeled radiopharmaceuticals for Positron Emission Tomography.</a> Applied radiation and isotopes. 121, 76-81 (2017).</li><li>Boudjemeline, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+solid+phase+supported+radiosynthesis+of+[(11)+C]PiB+using+tC18+cartridge+as+a+"3-in-1"+production+entity.">Highly efficient solid phase supported radiosynthesis of [(11) C]PiB using tC18 cartridge as a "3-in-1" production entity.</a> Journal of Labelled Compounds and Radiopharmaceuticals. 60 (14), 632-638 (2017).</li><li>Mathis, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lipophilic+thioflavin-T+derivative+for+positron+emission+tomography+(PET)+imaging+of+amyloid+in+brain.">A lipophilic thioflavin-T derivative for positron emission tomography (PET) imaging of amyloid in brain.</a> Bioorganic and medicinal chemistry letters. 12 (3), 295-298 (2002).</li><li>Mathis, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+11C-labeled+6-substituted+2-arylbenzothiazoles+as+amyloid+imaging+agents.">Synthesis and evaluation of 11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents.</a> Journal of medicinal chemistry. 46 (13), 2740-2754 (2003).</li><li>. Buffer Reference Center Available from: <a target="_blank" href="https://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html">https://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html</a> (2019)</li><li>Philippe, C., Mitterhauser, M., Wadsak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18,+Synthesis+of+2-(4-N-[11C]Methylaminophenyl)-6-Hydroxybenzothiazole+([11C]6-OH-BTA-1;+[11C]PIB).">Chapter 18, Synthesis of 2-(4-N-[11C]Methylaminophenyl)-6-Hydroxybenzothiazole ([11C]6-OH-BTA-1; [11C]PIB).</a> Radiochemical Syntheses. , 177-189 (2012).</li><li>Shao, X., Fawaz, M. V., Jang, K., Scott, P. J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Applications+of+[11C]Hydrogen+Cyanide.">Synthesis and Applications of [11C]Hydrogen Cyanide.</a> Radiochemical Syntheses. , 207-232 (2015).</li><li>Ametamey, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosynthesis+and+preclinical+evaluation+of+11C-ABP688+as+a+probe+for+imaging+the+metabotropic+glutamate+receptor+subtype+5.">Radiosynthesis and preclinical evaluation of 11C-ABP688 as a probe for imaging the metabotropic glutamate receptor subtype 5.</a> Journal of Nuclear Medicine. 47 (4), 698-705 (2006).</li><li>Ametamey, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+PET+studies+of+metabotropic+glutamate+receptor+subtype+5+with+11C-ABP688.">Human PET studies of metabotropic glutamate receptor subtype 5 with 11C-ABP688.</a> Journal of Nuclear Medicine. 48 (2), 247-252 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Despite the recent emergence and FDA approval of several 18F-labeled PET tracers, such as florbetapir, florbetaben and flutemetamol, [11C]PiB remains a gold standard tracer for amyloid imaging due to the fast brain uptake and low non-specific binding. Currently this tracer is synthesized via either wet chemistry16 or using a &#34;dry loop&#34; approach4,17. Both methods require HPLC purification followed by r...

Positron emission tomography (PET) is a molecular imaging modality which relies on detecting the radioactive decay of an isotope attached to a biologically active molecule to enable the in vivo visualization of biochemical processes, signals and transformations. Carbon-11 (t1/2 = 20.3 min) is one of the most commonly used radioisotopes in PET because of its abundance in organic molecules and short half-life which allows for multiple tracer administrations on the same day to the same human or animal subject and reduces the radiation burden on the patients. Many tracers labeled with this isotope are used in clinical studies and in basic health research for in vivo PET imaging of classical and emerging biologically relevant targets - [11C]raclopride for D2/D3 receptors, [11C]PiB for amyloid plaques, [11C]PBR28 for translocator protein - to name just a few.
Carbon-11 labeled PET tracers are predominantly produced via 11C-methylation of non-radioactive precursors containing -OH (alcohol, phenol and carboxylic acid), -NH (amine and amide) or -SH (thiol) groups. Briefly, the isotope is generated in the gas target of a cyclotron via a 14N(p,&#945;)11C nuclear reaction in the chemical form of [11C]CO2. The latter is then converted into [11C]methyl iodide ([11C]CH3I) via either wet chemistry (reduction to [11C]CH3OH with LiAlH4 followed by quenching with HI)1 or dry chemistry (catalytic reduction to [11C]CH4 followed by radical iodination with molecular I2)2. [11C]CH3I can then be further converted to the more reactive 11C-methyl triflate ([11C]CH3OTf) by passing it over a silver triflate column3. The 11C-methylation is then performed by either bubbling the radioactive gas into a solution of non-radioactive precursor in organic solvent or via the more elegant captive solvent &#34;loop&#34; method4,5. The 11C-tracer is then purified by means of HPLC, reformulated in a biocompatible solvent, and passed through a sterile filter before being administered to human subjects. All of these manipulations must be fast and reliable given the short half-life of carbon-11. However, the use of an HPLC system significantly increases the losses of the tracer and production time, often necessitates the use of toxic solvents, complicates automation and occasionally leads to failed syntheses. Furthermore, the required cleaning of the reactors and HPLC column prolongs delays between the syntheses of subsequent tracer batches and increases the exposure of personnel to radiation.
The radiochemistry of fluorine-18 (t1/2 = 109.7 min), the other widely used PET isotope, has been recently advanced via the development of cassette-based kits that obviate the need for HPLC purification. By employing solid phase extraction (SPE) cartridges, these fully disposable kits allow for the reliable routine production of 18F-tracers, including [18F]FDG, [18F]FMISO, [18F]FMC and others, with shorter synthesis times, reduced personnel involvement and minimal maintenance of the equipment. One of the reasons carbon-11 remains a less popular isotope in PET imaging is a lack of similar kits for the routine production of 11C-tracers. Their development would significantly improve synthetic reliability, increase radiochemical yields and simplify automation and preventive maintenance of the production modules.
Currently available production kits take advantage of inexpensive, disposable, SPE cartridges instead of HPLC columns for the separation of the radiotracer from unreacted radioactive isotope, precursor and other radioactive and non-radioactive by-products. Ideally, the radiolabeling reaction also proceeds on the same cartridge; for example, the [18F]fluoromethylation of dimethylaminoethanol with gaseous [18F]CH2BrF in the production of prostate cancer imaging PET tracer [18F]fluoromethylcholine occurs on a cation-exchange resin cartridge6. Although similar procedures for the radiolabeling of several 11C-tracers on cartridges have been reported7,8 and became especially powerful for the radiosynthesis of [11C]choline9 and [11C]methionine10, these examples remain limited to oncological PET tracers where the separation from the precursor is often not required. We recently reported the development of &#34;[11C]kits&#34; for the production of [11C]CH3I11 and subsequent 11C-methylation, as well as solid phase-supported synthesis12 in our endeavours to simplify the routine production of 11C-tracers. Here, we wish to demonstrate our progress using the example of the solid phase supported radiosynthesis of [11C]PiB, a radiotracer for A&#946; imaging which revolutionized the field of Alzheimer&#39;s disease (AD) imaging when it was first developed in 2003 (Figure 1)13,14. In this method, volatile [11C]CH3OTf (bp 100 &#176;C) is passed over 6-OH-BTA-0 precursor deposited on the resin of a disposable cartridge. PET tracer [11C]PiB is then separated from the precursor and radioactive impurities by elution from the cartridge with biocompatible aqueous ethanol. Further, we automated this method of [11C]PiB radiosynthesis using a remotely operated radiochemistry synthesis module and disposable cassette kits. Specifically, we implemented this radiosynthesis on a 20-valve radiochemistry module, equipped with syringe drive (dispenser) which fits standard 20 mL disposable plastic syringe, gas flow controller, vacuum pump and gauge. Due to the simplicity of this method, we are confident that it can be modified to most commercially available automated synthesizers, either cassette-based or those equipped with stationary valves. This solid phase supported technique facilitates [11C]PiB production compliant with Good Manufacturing Practice (GMP) regulations and improves synthesis reliability. The technique described here also reduces the amount of precursor required for radiosynthesis, uses only &#34;green&#34; biocompatible solvents and decreases the time between consecutive production batches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-OH-BTA-0</td>
			<td>ABX advanced biochemical compounds</td>
			<td>5101</td>
			<td>Non-radioactive precursor of [11C]PiB</td>
		</tr>
		<tr>
			<td>6-OH-BTA-1</td>
			<td>ABX advanced biochemical compounds</td>
			<td>5140</td>
			<td>Non-radioactive standard of [11C]PiB</td>
		</tr>
		<tr>
			<td>Agilent 1200 HPLC system</td>
			<td>Agilent</td>
			<td>Agilent 1200</td>
			<td>Analytical HPLC system</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Commercial alcohols</td>
			<td>432526</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (luer-tip, 250 &#181;L)</td>
			<td>Hamilton</td>
			<td>HAM80701</td>
			<td></td>
		</tr>
		<tr>
			<td>MZ Analytical PerfectSil 120</td>
			<td>MZ-Analysentechik GmbH</td>
			<td>MZ1440-100040</td>
			<td>Analytical HPLC column</td>
		</tr>
		<tr>
			<td>Perkin Elmer Clarus 480 GC system</td>
			<td>Perkin Elmer</td>
			<td>Clarus 480</td>
			<td>Gas chromotograph</td>
		</tr>
		<tr>
			<td>polycarbonate manifold</td>
			<td>Scintomics</td>
			<td>ACC-101</td>
			<td>Synthesis manifold</td>
		</tr>
		<tr>
			<td>Restek MTX-Wax column (30 m, 0.53 mm)</td>
			<td>Restek</td>
			<td>70625-273</td>
			<td>Analytical GC column</td>
		</tr>
		<tr>
			<td>Scintomics GRP module</td>
			<td>Scintomics</td>
			<td>Scintomics GRP</td>
			<td>Automated synthesis unit</td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 Plus</td>
			<td>Waters</td>
			<td>WAT020515</td>
			<td>Solid phase extraction cartridge</td>
		</tr>
		<tr>
			<td>solvent-resistant manifold</td>
			<td>Scintomics</td>
			<td>ACC-201</td>
			<td>Synthesis manifold</td>
		</tr>
		<tr>
			<td>Spinal needle</td>
			<td>BD</td>
			<td>405181</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile extension line</td>
			<td>B. Braun</td>
			<td>8255059</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filter</td>
			<td>Millipore</td>
			<td>SLLG013SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile vial (20mL)</td>
			<td>Huayi</td>
			<td>SVV-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Baxter</td>
			<td>JF7623</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthra MeIplus Research</td>
			<td>Synthra</td>
			<td>MeIplus Research</td>
			<td>[11C]CH3I/[11C]CH3OTf module</td>
		</tr>
		<tr>
			<td>Syringe (10 mL)</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (1mL)</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (20 mL)</td>
			<td>B. Braun</td>
			<td>4617207V</td>
			<td>Dispenser syringe</td>
		</tr>
		<tr>
			<td>Vent filter</td>
			<td>Millipore</td>
			<td>TEFG02525</td>
			<td></td>
		</tr>
	</tbody>
</table>

solid phase 11c-methylation, purification and formulation for the production of pet tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

To summarize a typical radiosynthesis of [11C]PiB, gaseous [11C]CH3OTf is first passed through a tC18 cartridge preloaded with a solution of precursor (Figure 1). Separation of the reaction mixture is then achieved by successive elution with aqueous ethanol solutions as follows. First, 12.5% EtOH elutes the majority of unreacted [11C]CH3OTf and 6-OH-BTA-0, then 15% EtOH washes out the residual impurities,...

Watch this Scientific Journal Video about Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers at JoVE.com

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

solid-phase-11c-methylation-purification-formulation-for-production

Research

JoVE Journal

Chemistry

6.5K Views.  McGill University. We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Video: Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Supercritical Nitrogen Processing for the Purification of Reactive Porous Materials

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. Herein, nitrogen is presented as a novel supercritical drying fluid for specialized applications such as in the processing of reactive porous materials, where carbon dioxide and other fluids are not appropriate due to their higher chemical reactivity. Nitrogen exhibits similar physical properties in the near-critical region of its phase diagram as compared to carbon dioxide: a widely tunable density up to ~1 g ml-1, modest critical pressure (3.4 MPa), and small molecular diameter of ~3.6 &#197;. The key to achieving a high solvation power of nitrogen is to apply a processing temperature in the range of 80-150 K, where the density of nitrogen is an order of magnitude higher than at similar pressures near ambient temperature. The detailed solvation properties of nitrogen, and especially its selectivity, across a wide range of common target species of extraction still require further investigation. Herein we describe a protocol for the supercritical nitrogen processing of porous magnesium borohydride.

Nitrogen is an effective supercritical fluid for extraction or drying processes due to its small molecular size, high density in the near-liquid supercritical regime, and chemical inertness. We present a supercritical nitrogen drying protocol for the purification treatment of reactive, porous materials.

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2&#039;-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...