Establishment of a candidate reference method and verification of reference intervals for serum copper in pregnant and non-pregnant women

Background: Serum copper is an essential trace element, and accurate quantification is important for assessing copper status in pregnancy. Current serum copper assays are limited by inaccuracy, poor inter-method comparability, and difficulties in transferring reference intervals (RIs) between populations. This study aimed to establish an inductively coupled plasma mass spectrometry (ICP-MS)-based candidate reference method for serum copper and to verify whether multicenter RIs for serum copper can be transferred to a Chinese population. Methods: Cobalt was used as the internal standard. After nitration with ultrapure nitric acid, samples were diluted 100-fold with ultrapure water. The ⁶³Cu/⁵⁹Co ratio was measured in helium collision mode at a gas flow rate of 5 mL/min. Analytical performance was validated, and serum copper RIs in pregnant and non-pregnant women were subsequently verified. Results: The candidate reference method demonstrated excellent linearity, with a correlation coefficient (R) >0.99998 over 0–13.75 μg/g. Total precision ranged from 0.30% to 0.40%, and spike recovery was 100.06% (99.95–100.14%). Trueness was confirmed by measurements of NIST SRM 1598a within the certified value and stated uncertainty. The RI for healthy non-pregnant women was transferable to healthy Chinese women, whereas the pregnancy-specific RI was not fully transferable. Conclusion: A highly sensitive and specific candidate reference method for serum copper was established, strengthening the reference measurement system and contributing to the standardization and harmonization of serum copper assays. Pregnancy-specific RIs for serum copper in Chinese women should be established.