High sensitivity trace-metal detection using an HPLC-based assay

Objectives Measurement of trace metal contamination is critical to the development of new methods of radiometal production and their application in labeling biomolecules of clinical interest. The widely used radiometal ⁶⁴Cu is typically produced at a specific activity of 200 μCi/μg, which at a concentration of 2 mCi/μL is equivalent to 10 ppm Cu. Ideally, this information should be available using an inexpensive, reproducible assay that can be carried out using commonly available instrumentation. ICP-MS provides these data with high sensitivity and specificity, but at high cost, limiting access. TETA titration provides high sensitivity and low cost but with the disadvantage of low specificity. In this study, we developed a new method to obtain these data with high sensitivity but at relatively low cost based on an HPLC using fluorescence detection.

Methods The bifunctional chelator p-NH₂-Bn-DOTA was conjugated with fluorescein isothiocyanate (FITC) and the resulting fluorescein-DOTA (FD) conjugate was purified by HPLC. The FD conjugate was incubated for >1h at room temperature in acetate buffer (0.1 M, pH 5) with copper(II) samples at concentrations as low as 10 parts per billion (ppb). The fluorescently tagged Cu-DOTA chelate was analyzed using reverse-phase HPLC (mobile phase: Solvent A; 3-(N-morpholino)propanesulfonic acid, 0.05 M, pH 7: Solvent B; acetonitrile; gradient 5% solvent B at 5 minutes to 30% solvent B at 20 minutes).

Results Copper complexation by FD increased the lipophilicity of the Cu(II) (t(r) = 13.7 min) allowing it to be separated from uncomplexed FD (t(r) = 12.9 min). At 10 ppb Cu(II), the signal to noise ratio was much greater than 5, indicating that this was a valid observation.

Conclusions Using this simple and relatively inexpensive method, it is possible to detect Cu at concentrations as low as 10 ppb, more than an order of magnitude more sensitive than required for the measurement of non-radioactive Cu in typical samples of Cu-64.

Research Support Children's Hospital Radiology Foundation.