A Sensitive Method for the Measurement of Copper at Trace Levels Using an HPLC-Based Assay.

Background: Measurement of trace metal contamination is critical in the production of radiometals, such as 64Cu, for protein labeling. ICP-MS provides these data with high sensitivity and high specificity, but at high (instrument) cost. TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid) titration provides high sensitivity at low cost but with low specificity. A method that allowed the measurement of trace metals with high sensitivity but also at relatively low cost would, therefore, be very useful in the development of new radiometal production methods.

Objective: The goal of this project was to develop an analytical method for copper that uses readily available laboratory equipment while minimally achieving low ppm sensitivity.

Method: The metal-chelating macrocycle 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA) was coupled to fluorescein to produce a molecule that combines high UV absorbance and high quantum yield with the ability to chelate a wide range of transition metals. The fluorescein-DOTA was mixed with Cu(II) samples at low ppm concentrations, and the samples were analyzed by reversed-phase HPLC.

Results: Copper chelation by the DOTA moiety decreased its overall charge, leading to a delayed elution from a C18 HPLC column. The absorbance signal of the fluorescein-DOTA-Cu(II) peak (453 nm) linearly correlated with the copper concentration allowing measurement of Cu(II) down to 1.25 ppm. Furthermore, using fluorescence detection (521 nm) the detection limit was reduced by almost three orders of magnitude, to 2.5 ppb (p<0.05).

Conclusion: Using a fluorescent dye (fluorescein) coupled to a macrocyclic chelator (DOTA) and an HPLC equipped with a standard UV detector is it possible to measure Cu at ppm concentrations, the Cu concentration observed in typical samples of 64Cu.