A new anion exchange purification method for Cu stable isotopes in blood samples

The isotopic composition of iron, zinc, copper, and cadmium (δ 56 Fe, δ 66 Zn, δ 65 Cu, and δ 114 Cd) are novel and promising tools to study the metabolism and homeostasis of trace metals in the human body. Serum δ 65 Cu has been proposed as a potential tool for diagnosis of cancer in liquid biopsy, and other metals may have similar utility. However, accurate analysis of trace metal isotopes is challenging because of the difficulties in purifying the metals from biological samples. Here we developed a simple and rapid method for sequential purification of Cu, Fe, Zn, and Cd from a single blood plasma sample. By using a combination of 11 M acetic acid and 4 M HCl in the first steps of column chemistry on AG-MP1 resin, we dramatically improve the separation of Cu from matrix elements compared to previous methods which use concentrated HCl alone. Our new method achieves full recovery of Cu, Fe, Zn, and Cd to prevent column-induced isotope fractionation effects, and effectively separates analytes from the matrix in order to reduce polyatomic interferences during isotope analysis. Our methods were verified by the analysis of isotope standards, a whole blood reference material, and a preliminary sample set including five plasma samples from healthy individuals and five plasma samples from cancer patients. This new method simplifies preparation of blood samples for metal isotope analysis, accelerating multi-isotope approaches to medical studies and contributing to our understanding of the cycling of Fe, Zn, Cu, and Cd in the human body. Graphical abstract ᅟ