Development and evaluation of a rapid analysis for HEPES determination in 68Ga-radiotracers

Abstract

BackgroundHEPES is a favorable buffer for 68Ga-complexations in radiochemical laboratories. The drawback of this buffer is its prescribed limit of 200 μg per recommended application volume in the final formulation. Currently, a TLC test according to the European Pharmacopoeia (Ph. Eur.) has to be performed for quantification, but this analysis suffers from low reliability and reproducibility and is based on a subjective, semi-quantitative visual evaluation. In this study, the TLC method according to the Ph. Eur. and two literature-known HPLC assays for HEPES quantification were evaluated. Additionally, the development of an improved TLC method was performed.ResultsThe assay according to Antunes et al. provided a reasonable quantification of HEPES using HPLC. Additionally, a reliable and conclusive TLC method was developed, which facilitates quantitative analysis by means of a pixel-based evaluation. A comparison of those two methods with the Ph. Eur. TLC assay pinpoints the superiority of the HPLC as well as the new TLC assay. Furthermore, evaluation of HEPES contents using both TLC assays by 28 subjects supported the conclusion that the newly developed TLC method is clearly favorable.ConclusionThe TLC method according to the Ph. Eur. provides unsatisfactory results in terms of conclusiveness and reproducibility. In contrast, a reported HPLC assay showed valid results, with the drawback of high technical effort. An optimized alternative is provided by the improved TLC method described in this work that results in reliable outcomes and additionally offers quantitative analysis.

New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals

“…In routine, in principle, a limit test to determine if the HEPES content is below the maximum limit would be sufficient. Thus, the more reliable TLC method developed by Pfaff et al could be used in any nuclear medicine center (Pfaff et al 2018). However, even this method may sometimes give an overestimation of the HEPES amounts present in a sample, risking the non-release of the radiopharmaceutical.…”

Automated radiosynthesis, quality control, and biodistribution of Ga-68 pentixafor: First Indian experience

“…(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES is a Zwitterionic buffering agent of choice for 68Ga-Complexations but the only limitation is of the concentration of buffer (200.0 μg) in the final injection volume. [ 11 12 ] Hence, we standardized the volume of HEPES to be used to dilute the peptide. A standard of 3 ml of 1.5M HEPES buffer is available as a part of the standard reagent kit.…”

The use of HEPES-buffer in the production of gallium-68 radiopharmaceuticals – time to reconsider strict pharmacopoeial limits?

“…The European Pharmacopoeia prescribes a strict limit of 200 μg per dose for the HEPES content in radiopharmaceuticals intended for intravenous administration (European Pharmacopeia 2017 ). However, the method described in the European Pharmacopoeia to test for HEPES in radiopharmaceutical preparations has proven to be unreliable (Antunes et al 2020 ; Pfaff et al 2018 ; Sasson et al 2010 ). The low limit has hampered the use of HEPES in gallium-68 radiopharmaceuticals, despite its superior buffering properties.…”

“…Velikyan et al illustrated the beneficial effect of HEPES on specific activity in radiolabelling bioconjugates (Velikyan et al 2004). A number of references indicate that adjusting the pH with HEPES during radiolabelling with gallium-68 provides optimum results in terms of molecular activity, reproducibility, reliability and versatility (Bauwens et al 2010;Eppard et al 2014;Pfaff et al 2018;Velikyan et al 2004). The European Pharmacopoeia prescribes a strict limit of 200 μg per dose for the HEPES content in radiopharmaceuticals intended for intravenous administration (European Pharmacopeia 2017).…”