Serum copper is an essential trace element, and accurate quantification is important for assessing copper status in pregnancy and supporting early clinical interventions. However, current serum copper assays face several challenges, including potential inaccuracy, poor comparability between methods, and difficulties in transferring reference intervals (RIs) between populations. This study aimed to establish a simple inductively coupled plasma mass spectrometry (ICP-MS)-based candidate reference method for serum copper to strengthen the reference measurement system, and to verify whether recently proposed multicenter RIs for serum copper can be transferred to a Chinese population.
Methods
Cobalt was used as the internal standard. After digestion with Optima-grade ultrapure nitric acid, samples were diluted 100-fold with ultrapure water. The 63Cu/59Co ratio was measured in helium collision mode, with the gas flow rate set to 5 mL/min. Analytical performance was validated, and serum copper RIs in pregnant and non-pregnant women were subsequently verified.
Results
A candidate reference method was established. The method demonstrated excellent linearity, with a correlation coefficient (R) exceeding 0.99998 over the range 0–13.75 μg/g. The total precision ranged from 0.30% to 0.40%. The spike recovery was 100.06% (99.95–100.14%). Trueness was confirmed by measurements of NIST SRM 1598a, which fell within the certified value and its stated uncertainty. The RI for healthy non-pregnant women was transferable to healthy Chinese women, whereas the pregnancy-specific RI for serum copper was not fully transferable and requires further adjustment for Chinese women.
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.
SchwartzDAGrahamAL. The effects of pregnancy on women with COVID-19: maternal and infant outcomes. Clin Infect Dis2020; 71(16): 2042–2044. https://doi.org/10.1093/cid/ciaa559
CastrogiovanniPImbesiR. The role of malnutrition during pregnancy and its effects on brain and skeletal muscle postnatal development. J Funct Morphol Kinesiol2017; 2(3): 30. https://doi.org/10.3390/jfmk2030030
TokAÖzerAAlkan BaylanF, et al.Copper/zinc ratio can be a marker to diagnose ectopic pregnancy and is associated with the oxidative stress status of ectopic pregnancy cases. Biol Trace Elem Res2021; 199(6): 2096–2103. https://doi.org/10.1007/s12011-020-02327-0
6.
GuanLWangYLinL, et al.Variations in blood copper and possible mechanisms during pregnancy. Biol Trace Elem Res2024; 202(2): 429–441. https://doi.org/10.1007/s12011-023-03716-x
7.
PathakPKapilU. Role of trace elements zinc, copper and magnesium during pregnancy and its outcome. Indian J Pediatr2004; 71: 1003–1005. https://doi.org/10.1007/BF02828116
8.
RanjkeshFJalisehHKAbutorabiS. Monitoring the copper content of serum and urine in pregnancies complicated by preeclampsia. Biol Trace Elem Res2011; 144: 58–62. https://doi.org/10.1007/s12011-011-9026-9
9.
HaoYPangYYanH, et al.Association of maternal serum copper during early pregnancy with the risk of spontaneous preterm birth: a nested case-control study in China. Environ Int2019; 122: 237–243. https://doi.org/10.1016/j.envint.2018.11.009
10.
KeenCLUriu-HareJYHawkSN, et al.Effect of copper deficiency on prenatal development and pregnancy outcome. Am J Clin Nutr1998; 67(5): S1003–S1011. https://doi.org/10.1093/ajcn/67.5.1003S
11.
SkalnayaMGTinkovAALobanovaYN, et al.Serum levels of copper, iron, and manganese in women with pregnancy, miscarriage, and primary infertility. J Trace Elem Med Biol2019; 56: 124–130. https://doi.org/10.1016/j.jtemb.2019.08.009
12.
TsakiridisIKasapidouEDagklisT, et al.Nutrition in pregnancy: a comparative review of major guidelines. Obstet Gynecol Surv2020; 75(11): 692–702. https://doi.org/10.1097/OGX.0000000000000836
13.
ShenPJGongBXuFY, et al.Four trace elements in pregnant women and their relationships with adverse pregnancy outcomes. Eur Rev Med Pharmacol Sci2015; 19(24): 4690–4697.
14.
AndrewDGailRMoragB, et al.Recommended reference intervals for copper and zinc in serum using the US National health and nutrition examination surveys (NHANES) data. Clin Chim Acta2023; 546: 117397. https://doi.org/10.1016/j.cca.2023.117397
15.
LiuXZhangYPiaoJ, et al.Reference values of 14 serum trace elements for pregnant Chinese women: a cross-sectional study in the China nutrition and health survey 2010-2012. Nutrients2017; 9(3): 309. https://doi.org/10.3390/nu9030309
16.
BrownRJCMiltonMJT. Analytical techniques for trace element analysis: an overview. TrAC, Trends Anal Chem2005; 24(3): 266–274. https://doi.org/10.1016/j.trac.2004.11.010
17.
ArnaudJChappuisPZawislakR, et al.Comparison of serum copper determination by colorimetric and atomic absorption spectrometric methods in seven different laboratories. The S.F.B.C. trace element group. Clin Biochem1993; 26(1): 43–49. https://doi.org/10.1016/0009-9120(93)90016-y
HandleySAWanandyTPrenticeL. Validation of the randox colorimetric assays for serum copper and zinc. Ann Clin Biochem2024; 61(3): 182–194. https://doi.org/10.1177/00045632231208337
International Organization for Standardization (ISO). ISO 15195:2018. Laboratory medicine - requirements for the competence of calibration laboratories using reference measurement procedures. International Organization for Standardization, 2018.
22.
International Organization for Standardization (ISO). ISO 15193:2009. In vitro diagnostic medical devices - measurement of quantities in samples of biological origin - requirements for content and presentation of reference measurement procedures. International Organization for Standardization, 2009.
23.
Clinical and Laboratory Standards Institute (CLSI). EP28-A3c: defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline. 3rd ed.CLSI, 2010.
24.
LiuAXuPGongC, et al.High serum concentration of selenium, but not calcium, cobalt, copper, iron, and magnesium, increased the risk of both hyperglycemia and dyslipidemia in adults: a health examination center based cross-sectional study. J Trace Elem Med Biol2020; 59: 126470. https://doi.org/10.1016/j.jtemb.2020.126470
25.
MuñizCSFernández-MartinJLMarchante-GayónJM, et al.Reference values for trace and ultratrace elements in human serum determined by double-focusing ICP-MS. Biol Trace Elem Res2001; 82(1-3): 259–272. https://doi.org/10.1385/BTER:82:1-3:259
26.
GreenbergRRZeislerRKingstonHM, et al.Neutron activation analysis of the NIST bovine serum standard reference material using chemical separations. Fresenius Z Anal Chem1988; 332(6): 652–656. https://doi.org/10.1007/BF00472662
27.
Joint Committee for Traceability in Laboratory Medicine (JCTLM). CYC_II_L_I_RMP_21: NIST radiochemical neutron activation analysis method for copper via DDC extraction in chloroform. JCTLM database: higher-order reference materials, methods and services. Bureau International des Poids et Mesures (BIPM). [cited 2026 Jan 29]. Available at: https://www.jctlmdb.org/index/cyc_ii_l_i_rmp_21.html
28.
MunizCSMarchante GayonJMGarcia AlonsoJI, et al.Accurate determination of iron, copper and zinc in human serum by isotope dilution analysis using double focusing ICP-MS. J Anal At Spectrom1999; 14(9): 1505–1510. https://doi.org/10.1039/A901751H
29.
LauwensSCostas-RodríguezMVan VlierbergheH, et al.High-precision isotopic analysis of Cu in blood serum via multi-collector ICP-mass spectrometry for clinical investigation: steps towards improved robustness and higher sample throughput. J Anal At Spectrom2017; 32(3): 597–608. https://doi.org/10.1039/C6JA00433D
30.
HossainMKarmakarDBegumSN, et al.Recent trends in the analysis of trace elements in the field of environmental research: a review. Microchem J2021; 165: 106086. https://doi.org/10.1016/j.microc.2021.106086
MeyerSMarkovaMPohlG, et al.Development, validation and application of an ICP-MS/MS method to quantify minerals and (ultra-)trace elements in human serum. J Trace Elem Med Biol2018; 49: 157–163. https://doi.org/10.1016/j.jtemb.2018.05.012
33.
ChoiRSunJYooH, et al.A prospective study of serum trace elements in healthy Korean pregnant women. Nutrients2016; 8(11): 749. https://doi.org/10.3390/nu8110749
34.
DemirelMSelekSYildizT, et al.Determination of serum reference intervals for zinc and copper in adult populations in Malatya. Romanian J of Diab Nutr and Metab Dis2023; 30(1): 42–47. https://doi.org/10.46389/rjd-2023-1228
35.
TabriziFMPakdelFG. Serum level of some minerals during three trimesters of pregnancy in Iranian women and their newborns: a longitudinal study. Indian J Clin Biochem2014; 29(2): 174–180. https://doi.org/10.1007/s12291-013-0336-x
36.
KeshavarzPNobakhtMGBFMirhafezSR, et al.Alterations in lipid profile, zinc and copper levels and superoxide dismutase activities in normal pregnancy and preeclampsia. Am J Med Sci2017; 353(6): 552–558. https://doi.org/10.1016/j.amjms.2017.03.022
37.
ZhengYZhangCWeisskopfMG, et al.A prospective study of early pregnancy essential metal(loid)s and glucose levels late in the second trimester. J Clin Endocrinol Metab2019; 104(10): 4295–4303. https://doi.org/10.1210/jc.2019-00109
