HILIC - A simple method to determine [18F]fluoride in plasma and tissue samples

Objectives: The investigation of the metabolism of 18F-labeled radiotracers is a crucial step in the development process. Most often radio-defluorination as well as O- and N-dealkylation are observed resulting in the formation of very polar radiometabolites such as [18F]fluoride, [18F]fluoroethanol or [18F]fluoroacetic acid. Due to their high polarity these compounds are not easy to separate and analyze by the routinely used reversed phase HPLC. In a recent study we were faced with the problem of finding a high fraction of a very polar radiometabolite in brain samples of mice obtained after injection of a newly developed radiotracer, which seemed to be [18F]fluoride. However, as [18F]fluoride is not able to penetrate the blood-brain barrier to a considerable amount, we were interested to investigate the identity of this radiometabolite in the brain samples.

Methods: Hydrophilic interaction chromatography (HILIC) was selected as HPLC method since the elution order is more or less the reverse of the elution order in RP-HPLC. This is achieved by the use of a polar stationary phase and an aqueous mobile phase with a high content of organic solvent. In the present study, the retention and elution profile of [18F]fluoride was investigated by using a Nucleodur HILIC column (250 × 4.6 mm; Macherey-Nagel GmbH, Germany) and modifying the following parameters: i) organic solvent (e.g. CH3CN, CH3OH, THF), ii) pH value of the eluent, iii) concentration and nature of buffer additives (e.g. ammonium acetate, ammonium formate) and iv) isocratic and gradient mode. Typical investigated conditions comprised eluent compositions of 86-70% organic solvent/14-30% water, buffer concentrations ranging from 5 to 100 mM and acid concentrations of 0.05%.

Results:

The type of the organic solvent strongly influenced the retention of 18F]fluoride. Best results could be achieved with CH3CN, while with CH3OH almost no retention could be obtained. Also the pH value of the eluent played a crucial role. By using 0.05% trifluoroacetic acid (pH ~2) or formic acid (pH ~3) in the eluent, [18F]fluoride strongly retained on the stationary phase and eluted only at long retention times as very broad peak. The use of buffer systems at pH 6 to 8 improved the retention and the peak shape. Comparing ammonium formate and ammonium acetate the latter gave so far the best results. For example, under isocratic conditions at 74% CH3CN/20 mM NH4OAcaq, [18F]fluoride eluted at a retention time of 14 min while more lipophilic compounds such as typical 18F-labeled radiotracers elute close to the dead time of the column (A in Figure 1). Subsequently, this method was used to investigate homogenates of ex vivo brain samples of our newly developed P2Y1 receptor radiotracer [18F]1 (1-{2-[2-(tert-butyl)phenoxy]pyridin-3-yl}-3-[4-[18F.

Figure 1: A) HILIC radio-chromatogram of a mixture of [18F]1 and [18F]fluoride, B) HILIC radio-chromatogram of a mouse brain sample at 30 min p.i. of [18F]1; conditions: Nucleodur HILIC, 250 x 4.6 mm, 74% CH3CN/20 mM NH4OAcaq, flow 1.0 mL/min.

Conclusions:

Hydrophilic interaction chromatography is a simple and useful method to determine [18F]fluoride in biological samples such as plasma and brain homogenates. Further investigation is ongoing to further improve the retention profile and to use this method also for other typical polar radiometabolites such as [18F]fluoroacetic acid and [18F]fluoroethanol.

References: [1] Moldovan et al. Eur. J. Med. Chem. (in revision) "Studies towards the development of a PET radiotracer for imaging of the P2Y1 receptors in the brain: synthesis, 18F labeling and preliminary biological evaluation".