and the improved method when the sample solution is spotted next to the reference solution with the maximum concentration. Currently, a TLC test according to the European Pharmacopoeia (Ph. Cite this article. A new fast and reliable HPLC method to detect HEPES in [68Ga]-radiopharmaceuticals. Hence, we refined this method using the same composition of the matrix for both, the reference and the product solution, which resulted in spots with a similar shape. The latter contains the limiting HEPES concentration (which depends on the product activity as explained above) in water. Overall, the novel procedure offers a significant improvement to the available methods for HEPES testing. Velikyan I. CAS In contrast, a reported HPLC assay showed valid results, with the drawback of high technical effort. In parallel, the TLC method according to the Ph. In: Welch MJ, Redvanly CS, editors. Eur.) 1981;8(4):277–87. assay. Different mobile phases were tested to obtain an even more focused spot compared to the diffuse spot of the product solution according to the Ph. EJNMMI Res 8, 95 (2018). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (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, and indicate if changes were made. Correct assignments of HEPES concentration in comparison to the reference solution using visual inspection. Ingrid Leitinger and Friedrich Girschele are thanked for the routine 68Ga-radiopharmaceutical production and quality control. Andreas Krcal and Thomas Zenz are acknowledged for the technical support. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. However, a second HPLC run using the same HPLC system as for routine quality control requires proper pre-conditioning of the column after switching methods. https://doi.org/10.1186/s13550-018-0449-6, DOI: https://doi.org/10.1186/s13550-018-0449-6. Several spotting methods were tested (i.e., 8 × 1 μL, 4 × 2 μL, 8 × 2 μL, 5 × 5 μL, 8 × 5 μL), and results showed that spotting four times 2 μL gave intense spots after staining. Eur. Eur. 2010;10(16):1633–68. 7). was refined in order to obtain reliable and reproducible results, even when conducted by different operators. "Good buffers" such as HEPES are attributed with the following characteristics: HEPES is widely used in many biochemical reactions and as a buffering agent in some cell culture media. For a model calculation of a potential applicable patient dose of 150 MBq and an achieved product activity of 900 MBq in 16 mL, the maximum concentration of HEPES according to Ph. HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) is a commonly used zwitterionic buffer with a pKa of 7.3 at 37 °C. The product was tested for its HEPES content using HPLC and/or TLC aside from all other routine quality control tests. Calibration curves of HEPES concentration obtained from a pixel-based analysis method by means of ImageJ applied to the Ph. Cationic eluate pretreatment for automated synthesis of [68Ga]CPCR4.2. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid. The method for the Ph. Curr Top Med Chem. Eur. was valid in our laboratory setup and achieved an excellent linearity in a concentration range of 5–50 μg/mL HEPES (Fig. Representative HPLC chromatogram of HEPES (35 μg/mL) obtained by the method according to Antunes et al. Nevertheless, the latter one was reported to result in better radiochemical yields for [68Ga]Ga-DOTATOC than the first ones at low precursor amounts . Hence, HEPES should be the buffer of choice for clinical routine productions and especially scientific labeling approaches. The reaction mixture was heated to 126 °C for 6 min and subsequently purified applying a SPE cartridge (C-18, Sep-Pak Light). 8). The TLC assay according to the protocol of the Ph. SP, TN, VP, JC, and WW analyzed and interpreted the data. method was basically underestimated, which facilitated the release of radiopharmaceuticals that might fail the required quality criteria. Eur.) described this HPLC assay by applying an Agilent system. For evaluations of the TLC methods using ImageJ analysis, a calibration curve with an impressive R2 of 0.9956 was created for the newly developed TLC assay, in contrast to the calibration curve of the TLC method according to Ph. Contrast Media Mol Imaging. Further changes in the spotting method could facilitate the TLC assay. Evaluation of the stained TLC plates was additionally interpreted by means of a pixel-based analysis (using ImageJ software). Especially, Chrysoula Vraka is thanked for her continuing support. Eur.) Gallium-68 was eluted with NaCl solution (5 M) and transferred to a reactor containing either 40 μg Pentixafor or 2.5–10 μg PSMA-11 in 1.5 mL HEPES buffer (1 M). Sodium acetate (NaOAc), sodium succinate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were reported as the most favorable buffers for 68Ga-labeling so far . All authors read, revised, and approved the final manuscript. Eur. Representative images of iodine-stained TLC plates (35 μg/mL HEPES) with the sample (“P”) spotted in the left column and the maximum allowed concentration (“max,” 25 μg/mL in this experimental setup) in the right column of each plate. Google Scholar. 68Ga-based radiopharmaceuticals: production and application relationship. In this case, a recommended volume of V = 8 mL was arbitrarily chosen. Focusing on gallium-68, the pH must be in a range of 3.5 to 5.5 due to the fact that gallium starts to form insoluble colloids and hydroxides at a higher pH that are not available for complexation [2, 3]. A quantifiable HEPES peak was found applying a flow rate of 0.7 mL/min and a mobile phase of 15 mM ammonium formate at pH 9.5. gave different shapes of iodine spots of product samples (same formulation as the synthesized radiopharmaceutical) and the reference solution (in water). Division of Nuclear Medicine, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Waehringer Guertel 18-20, A-1090, Vienna, Austria, Sarah Pfaff, Tina Nehring, Verena Pichler, Jens Cardinale, Markus Mitterhauser, Marcus Hacker & Wolfgang Wadsak, Department of Inorganic Chemistry, University of Vienna, Vienna, Austria, Ludwig Boltzmann Institute Applied Diagnostics, Vienna, Austria, Tina Nehring, Jens Cardinale & Markus Mitterhauser, CBmed – Center for Biomarker Research in Medicine, Graz, Austria, You can also search for this author in In this article, the emphasis is put on one important parameter, the pH value that has to be taken into account, especially for complexations with radiometals. statement and 2011;23:310–3. provided a reasonable quantification of HEPES using HPLC. HEPES buffer, with pH values ranging from 6.8 - 8.2, can be prepared at a wide variety of ratios. Radiolabeling was performed according to a cationic purification protocol described elsewhere . The spotted volume of each solution has to be the recommended administration volume V (in mL) divided by 2000 and spotted in 1 μL steps. Correspondingly, pH buffering systems are necessary for a successful product formation.