With the aim of phasing out Class II solvents, as well as MEK, from our carbon-11 radiopharmaceutical production program for the reasons outlined above, we considered alternative solvents with which to conduct carbon-11 loop chemistry. Whilst there are many Class III solvents to choose from that could achieve this goal, it quickly became apparent that, even for quarterly residual solvent analyses, GC methods would have to be developed for residual solvent analysis of any solvent chosen, except perhaps ethanol. We routinely employ ethanol in HPLC mobile phases, as well as formulate radiopharmaceutical doses in 5 – 10% v/v ethanol, and so have reliable GC methods in place for its analysis.
[11C]DASB and [11C]raclopride were selected as the two radiopharmaceuticals for initial investigation of the ethanolic loop chemistry concept because, of all the radiopharmaceuticals labeled with carbon-11 using loop methods in this program, these two are by far the most commonly requested by nuclear medicine physicians at the University of Michigan. In initial attempts to prepare [11C]raclopride, 1 mg of TBA precursor 3 was dissolved in 100 µL of ethanol and loaded into the HPLC loop. [11C]MeOTf was then blown through the loop according to our standard protocol, 40 mL/min for 3 minutes. Analysis of the reaction mixture confirmed the production of [11C]raclopride, and the notion that loop syntheses can be conducted using ethanol. The combination of the low amount of solvent, nucleophilicity of the raclopride precursor TBA salt, and relatively high flow rate of the [11C]MeOTf combined to promote radiolabeling and minimize solvolysis of ethanol. As shown in the preparative HPLC trace (Figure 6), >85% of the radioactivity corresponded to [11C]raclopride, and radioactive byproducts were limited. [11C]Raclopride could then be purified using our standard, previously reported, semi-preparative HPLC method. Final dilution with saline provided formulated [11C]raclopride.
Similar results were obtained for the corresponding synthesis of [11C]DASB. In this case, the existing semi-preparative HPLC method (in which precursor eluted off first and occasionally tailed into the product peak) was replaced with the newly reported method from Gillings and colleagues, and a subsequent reconstitution into ethanolic saline. The new HPLC method is attractive as it actually reverses the elution order of DASB and the precursor, and so eliminates the chance of contamination resulting from precursor tailing into the product peak. This resulted in improved chemical purity of [11C]DASB (less minor impurities presumably associated with residual precursor) and, interestingly, also appears to be reflected in improved specific activity. Using the old HPLC method, the average specific activity for [11C]DASB was 5239 Ci/mmol (n = 10). Replacing the semi-preparative method with the new HPLC method resulted in 3-fold higher specific activities of 15152 Ci/mmol (n = 9).
At this stage, both [11C]raclopride and [11C]DASB were synthesized, purified and formulated using only ethanol. The only remaining solvent requiring elimination from the process was acetone, typically used during daily and weekly cleaning and drying of the synthesis module. Therefore the previously reported clean and dry cycles were modified to use only ethanol: clean (water), disinfect (70% ethanol) and dry (neat ethanol). Reflecting the decreased volatility of ethanol compared to acetone, the drying cycle was extended and the HPLC loop was blown dry with helium for 10 minutes, rather than the usual 5 minutes.
Following these initial positive results, simple proof-of-concept studies were demonstrated by preparing both radiopharmaceuticals multiple times. [11C]Raclopride was routinely obtained in 3.7% radiochemical yield (n = 64, based upon [11C]CO2), higher than yields for the corresponding loop methylation conducted in MEK previously disclosed by this group (2.2%, n = 10, based upon [11C]CO2). This increase in yield is attributed to improved solubility of the TBA salt of raclopride precursor in ethanol, versus MEK. Similarly, [11C]DASB was prepared in 3.0% radiochemical yield (n = 9, based upon [11C]CO2), comparable to previously reported yields obtained using MEK as the solvent (4.0%, n = 10, based upon [11C]CO2). Following purification and formulation, doses were submitted for QC testing (), which confirmed that the products were suitable for clinical use. Building on this, both of these synthesis methods are now employed to meet routine clinical supply of both [11C]raclopride and [11C]DASB.
Another aspect of the synthesis of [11C]DASB with scope for improvement is handling the radiolytic decomposition of the product. We have previously investigated radiolysis of radiopharmaceuticals, and demonstrated that many aniline-containing species, including [11C]DASB, are prone to radiolytic decomposition. Such decomposition results in radiochemical impurities in the final product, leading to batch failures because radiochemical purity (RCP) specifications are not met. Thus, when preparing [11C]DASB using traditional methods, it is essential to add sodium ascorbate to the final formulation to inhibit radiolysis. However, a drawback of adding sodium ascorbate to final formulations is that the UV signal associated with ascorbate is so large that it overwhelms the analytical HPLC trace (), making accurate analysis of cold [12C]DASB to determine specific activity, and any other chemical impurities in the dose, problematic. Trapping of [11C]DASB on the sep-pak is where the radiopharmaceutical is at its most concentrated, and we believe at its most vulnerable to radiolytic degradation. Considering this issue, we realized that the fairly high concentration of ethanol involved in reformulation, resulting from the change in HPLC mobile phase from our previous method (100 mM NH4OAc in 30% MeCN, pH = 5.5), to that reported by Gillings and co-workers (10 mM NH4OAc in 80% EtOH), might impart the same anti-oxidant protection previously imparted by sodium ascorbate during the critical reformulation step. Therefore, we repeated the synthesis of [11C]DASB without any sodium ascorbate and found that our supposition about the presence of ethanol was correct. Concentrations proved to offer sufficient anti-oxidant protection so that [11C]DASB was obtained in greater than 95% radiochemical purity (, n = 3). Moreover, doses were found to be stable up to 1 h after end-of-synthesis (data not shown), and HPLC analysis of levels of unlabeled [12C]DASB present in doses was greatly simplified because of the lack of an ascorbate peak in the analytical HPLC traces ().