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Iron, copper and zinc isotope compositions of

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Cite this: J. Anal. At. Spectrom., 2023,

biological reference materials determined by MC-
38, 2365 ICP-MS†
Rui Guo,a Hui-Min Yu, *ab Shu-Bin Fang,a Zi-Cong Xiaoa and Fang Huang ab

Received 18th August 2023
Accepted 2nd October 2023
DOI: 10.1039/d3ja00281k
rsc.li/jaas
The transition metals Fe, Cu and Zn are the key elements for life, and their isotope compositions are
expected to be a new index to estimate the trophic level of organisms and diagnose human diseases.
Therefore, it is important to analyse these isotopes precisely and accurately in biological samples with
low Fe, Cu and Zn abundances. In this study, we modify the one-column method to purify Cu–Fe–Zn
for isotope analysis. The parameters that could affect the accuracy and precision of isotopic
measurement were evaluated. The Fe, Cu and Zn isotope values of nine biological reference materials
are reported, including plants, marine and terrestrial animals, human hair and serum. The isotope
compositions of plant and marine animal organs are different from terrestrial animals and human organs.
The variation ranges of d56Fe, d65Cu and d66Zn from plant and marine animals are −1.23& to 0.11&,
0.39& to 0.53& and 0.28& to 0.87&, respectively, while those from terrestrial animals and human
tissues are −2.40& to 0.06&, −0.33& to 2.76& and −0.65& to 0.24&, respectively. This one-column
method is important to provide comprehensive isotope information for biological individuals and
efficient study Fe, Cu and Zn isotope distribution in paleontology and biomedicine fields.

widely used in archaeology and paleontology. They can help

1. Introduction
Because of the important catalytic, structural, and regulatory
roles, the transition metals (such as Fe, Cu and Zn) have been
identied as necessary micronutrients for life. In plants, Fe is an
essential element for the synthesis of iron–sulfur proteins and
a catalyst for enzyme-mediated redox reactions.1 In animals, it is
a basic component of heme, which is used to transport oxygen
and carbon dioxide in the body.2 Copper plays a major role in
oxidase to control electron transfer, which is important for
energy generation and brain activity in the animal body. When
Cu homeostasis is unregulated, especially when it presents in
excess, its oxidative potential can generate reactive free radicals
that contribute to cancers, nervous system disease and aging.3,4
Zinc is utilized as a cofactor in more than 300 enzymes that affect
a large variety of biological processes. Therefore, Fe, Cu and Zn
are also regarded as key elements for life.
Biological metabolism can cause signicant isotope frac-
tionation of Fe, Cu, and Zn between the organs of plants and
animals.2,5 Therefore, Fe, Cu, and Zn isotope compositions are
a
CAS Key Laboratory of Crust-Mantle Materials and Environments, School of Earth
and Space Sciences, University of Science and Technology of China, Hefei, Anhui
230026, China. E-mail: huy16@ustc.edu.cn
b
CAS Center for Excellence in Comparative Planetology, University of Science and
Technology of China, Hefei, Anhui 230026, China
† Electronic supplementary information (ESI) available. See DOI:
https://doi.org/10.1039/d3ja00281k
reconstruct ancient food chains.6–10 Jaouen et al.6 found that Fe,
Cu, and Zn isotope compositions varied signicantly between
herbivore bones and carnivore bones, which can be used to
distinguish animals with different trophic levels. Zn isotopes
have been widely applied to establish an equation for assessing
the trophic level of diverse extant and extinct sharks.10 Fe and
Cu isotopes were used to provide a criterion of sex for incom-
plete human remains, because men's bone is depleted in 56Fe
and enriched in 65Cu compared to women's bone.11
Some diseases disrupt biochemical pathways and homeo-
stasis associated with metalloproteins, and affect metal stable
isotopes such as Fe, Cu and Zn. Therefore, metal stable isotopes
can be used to study biomedical diagnosis, disease or metabo-
lism.2 Fe isotopes are fractionated during metabolism,
including Fe absorption by the intestine, storage in the liver and
the synthesis of hemoglobin and myoglobin.12,13 Therefore, Fe
isotopes (d56Fe) can trace the diseases affecting Fe metabolism,
such as thalassemia,14 chronic kidney disease,15 acute coronary
syndrome (ACD), and hemochromatosis.12 Cu isotope compo-
sitions (d65Cu) were used as tracers of Wilson's disease,16 and
were considered to be a diagnostic tool for cancer.17 An
increasing number of studies have reported Cu isotopes in the
tissues and serum of patients with different cancers, including
breast cancer, cervical cancer,18 colorectal cancer19 and liver
cancer.20,21 In those studies, compared with control samples,
cancer tissues had heavier Cu isotopes, and the serum of
patients with cancer had lighter Cu isotopes. Zn isotopes (d66Zn)

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can also provide additional supplementation for hemochro-
matosis.22 In addition, there are signicant Zn isotope varia-
tions between the patients with breast cancer or prostate cancer
and control samples,23 indicating that Zn isotopes can also be
used as cancer tracers. Fe, Cu and Zn isotopes in human serum
and human brain are proposed to help diagnose Alzheimer's
disease.24 Therefore, combining the isotope data of three
elements may provide more and complementary information
for disease diagnosis.
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It is a challenge to simultaneously analyse Fe, Cu and Zn
isotopes of biological samples, because these samples generally
have low concentrations of metal elements, high contents of
organic matter, and limited sample sizes (e.g., serum). Most
biological samples have heterogeneous isotope compositions. If
we analyse Fe, Cu and Zn isotopes of the sample aliquots with
separated digestion and purication processes, the isotope data
might be isolated between different aliquots of the same sample.
To obtain the comprehensive Fe, Cu and Zn isotope composi-
tions of biological samples precisely and accurately, we estab-
lished a one-column separation method to purify Fe, Cu and Zn
simultaneously from the same biological sample aliquot.
It is also necessary to have reference materials with compo-
sitions similar to biological samples.25 These reference mate-
rials should have well-characterized isotope compositions and
widely available sources. Up to now, the on-sale biological
reference materials with reported Fe, Cu and Zn isotope
compositions are very scarce. In this study, we report Fe, Cu and
Zn isotope data for nine biological reference materials, eight of
which are rst reported. These data could be benecial to future
biological or medical studies as a quality control.
2. Methods
2.1 Biological reference materials
This study analyses nine biological reference materials,
including three plants and six animals (including human serum
and hair). The three plant reference materials were from the
National Institute of Metrology of China (NIM), including
skimmed soybean (GBW10054), whole soybean (GBW10055),
and pumpkin (GBW(E)100199). The six animal reference mate-
rials were prawn (GBW10050) and pig liver (GBW10051) from the
Institute of Geophysical and Geochemical Exploration (IGGE) of
the Chinese Academy of Geological Sciences (CAGS), yolk
(GBW(E)100198) from NIM, human hair (GBW09101b) from the
Shanghai Institute of Applied Physics (SINAP) of the Chinese
Academy of Science (CAS), human serum (GBW(E)090933) from
the Beijing Institute of Medical Testing (BIMT), and pig kidney
(ERM-BB186) from the Institute of Reference Materials and
Measurement (IRMM) of European Commission (EC) Joint
Research Centre (JRC). Except for ERM-BB186, whose Fe, Cu and
Zn isotopes have been reported, all other eight biological refer-
ence materials were rst analysed for all Fe, Cu and Zn isotopes.
2.2 Sample digestion
The biological reference materials were analysed in the CAS Key
Laboratory of Crust-Mantle materials and Environments at the
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University of Science and Technology of China (USTC). Water
was puried to 18.2 MU cm by a Milli-Q Element system (Mil-
lipore Corporation, Burlington, Massachusetts, USA), and high-
purity TraceMetal™ grade acids (HNO3, HF and HCl, Thermo
Fisher Scientic, Waltham, MA, USA) were puried using
double sub-boiling distillation.
Approximately 100–600 mg of sample powder was weighed
into vessels (modied polytetrauoroethylene) with 4.8 mL
high-purity H2O2 and 1.6 mL concentrated HNO3. Aer holding
at room temperature for 24 h, the vessels were heated on
a hotplate at 100 °C for 24 h to remove partial organic matter.
Aer evaporating to dryness and treating with 1 mL concen-
trated HF, 1 mL concentrated HNO3, and 3 mL concentrated
HCl, the vessels were placed inside a microwave digestion
instrument. The instrument was run by ramping up the
temperature stepwise to 200 °C and 40 atm over 4 minutes, and
holding there for 30 minutes. Then, continued to heat up to
225 °C and 45 atm and held there for 30 minutes to ensure
complete digestion. Then, the digestion solution and rinsing
solution were transferred into 30 mL PFA screw cap beakers and
evaporated to dryness. Finally, the samples were dissolved in
1 mL 6 mol L−1 HCl for ion exchange separation.
2.3 Chemical purication
The one-column separation of Cu–Fe–Zn in biological samples
used in this study is modied from Maréchal et al.26 and Chen
et al.27 The 10 mL Bio-Rad Poly-Prep column with 2 mL anion-
exchange resin of AG MP-1M (100–200 mesh) was used to
separate different elements in the large sample size of biological
samples. The details of chemical purication are given in Fig. 1
and it will take 6 hours to separate Cu, Fe, and Zn. Most matrix
elements such as Na, Mg, Al, Ca and K were removed by 6 mL
6 mol L−1 HCl, and Cu, Fe and Zn were collected in the
following 26 mL 6 mol L−1 HCl, 6 mL 0.5 mol L−1 HCl and
13 mL mol L−1 HNO3, respectively (Fig. S1†). In this procedure,
0.5 mol L−1 HCl rather than 2 mol L−1 HCl was used to elute Fe,
since the adsorption of Fe on anionic resins decreases with
decreasing acidity of HCl.28 This modication effectively sepa-
rates Fe from other elements and reduces the dosage of acid.
For most biological samples, matrix elements can be effi-
ciently removed in the rst column separation. However, a few
samples may need two-steps to further purify Fe, Cu or Zn due
to their very low contents in these biological samples. For Cu,
the second column was the same as the rst column, which was
a 10 mL Bio-Rad Poly-Prep column with 2 mL of AG MP-1M
(100–200 mesh) anion-exchange resin; for Fe, the second
column was a 2 mL Bio-Rad Poly-Prep column with 0.5 mL of
AG1X8 (100–200 mesh) anion-exchange resin; for Zn, the second
column was a 2 mL Bio-Rad Poly-Prep column with 0.5 mL of AG
MP-1M (100–200 mesh) anion-exchange resin. The details are
listed in Fig. 1. The nal Cu, Fe and Zn cuts were evaporated to
dryness and conditioned for Multi-Collector Inductively
Coupled Plasma Mass Spectrometer (MC-ICP-MS) measurement
with 2 wt% HNO3. The recoveries of Fe, Cu and Zn were 94.7%,
93.0% and 99.9%, respectively. The total procedural blanks of
Fe and Zn were <20 ng and of Cu was <2 ng, which was
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Fig. 1 Schematic view of the procedures used for Fe, Cu and Zn isotope measurement.

negligible compared with the amount of the total loaded (Zn > 2
mg, Cu > 1 mg, and Fe > 5 mg).
2.4 Mass spectrometry
The isotope compositions of Fe, Cu and Zn were determined by
MC-ICP-MS (Thermo Fisher Scientic Neptune Plus, Bremen,
Germany) in the same laboratory. All instrumental parameters
are listed in Table 1. For Fe isotope analysis, sample solutions
were introduced using a quartz dual cyclonic spray chamber
(Thermo Fisher Scientic), a PFA microow nebulizer
(Elemental Scientic Inc, Omaha, NE, USA) with an uptake rate
of ∼50 mL min−1, nickel H skimmer cone and jet cone (Thermo
Fisher, Bremen). The instrumental mass bias of Fe isotope
ratios was corrected by the standard-sample-bracketing (SSB)
method,29 and IRMM 014 was used as the bracketing standard.
Four Fe isotopes (54Fe, 56Fe, 57Fe, 58Fe) were collected by L1, C,
H1 and H2, respectively, and 53Cr and 62Ni were collected in L3
and H4 Faraday cups to monitor their isobaric interference with
Fe isotopes. The instrumental sensitivity was ∼6 V (mg−1 g−1) for
56
Fe in high-resolution mode. Data of one block with 30 cycles
were collected, and the integration time was 4.194 s per cycle.
Before each measurement, the sample introduction system was
cleaned with 5 wt% HNO3 and 2 wt% HNO3 until the 56Fe signal
was less than 10 mV to eliminate the potential cross-
contamination.
For Cu isotope analysis, the instrumental parameters were
similar as Fe isotope analysis. The instrumental mass bias of Cu
isotope ratios was also corrected by SSB method, and ERM-
AE647 was used as the bracketing standard.30 The ion
currents of 63Cu and 65Cu were collected in L2 and C Faraday

Table 1 Instrumental operating parameters for Fe, Cu, and Zn isotope measurements

Neptune plus Fe isotopes Cu isotopes Zn isotopes

RF power 1200 W 1200 W 1200 W

Cooling Ar ∼16 L min−1 ∼16 L min−1 ∼16 L min−1
Auxiliary Ar ∼0.8 L min−1 ∼0.8 L min−1 ∼0.8 L min−1
Nebuliser Ar ∼0.85 L min−1 ∼1.0 L min−1 ∼0.85 L min−1
Extraction voltage −2000 V −2000 V −2000 V
Resolution High-resolution Low-resolution High-resolution
Integration time 4.194 s 4.194 s 4.194 s
Number of cycles 30 30 30
Typical sensitivity ∼6 V (mg−1 g−1) for 56Fe ∼35 V (mg−1 g−1) for ∼18 V (mg−1 g−1) for 64Zn
63
Cu
Cones H + Jet H + Jet H + Jet
Cup conguration L3 L1 C H1 H2 H4 L2 L1 C L4 L2 C H1 H3 H4
53 54 56 57 58 62 63 64 65 62 64 66 67 68 70
Cr Fe Fe Fe Fe Ni Cu Zn Cu Ni Zn Zn Zn Zn Zn

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cups, respectively, at a low resolution with the sensitivity of 35 V
(mg−1 g−1) for 63Cu. Prior to each measurement, the 63Cu signal
should be cleaned below 3 mV.
For Zn isotope analysis, the instrumental mass discrimina-
tion and chemical purication of Zn isotopes was correct by the
double-spike technique and 70Zn–67Zn was applied as the
double-spike pair.31 The sample solution was introduced by an
Aridus III desolator (CETAC Technologies, Omaha, NE, USA) in
a high resolution mode, and the sensitivity was 18 V (mg−1 g−1)
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for 64Zn. Potential cross-contamination was eliminated with 5
wt% HNO3 and 2 wt% HNO3 cleaning until the signal of 64Zn
was below 3 mV.
The isotope compositions of Fe, Cu and Zn are denoted by
d notation as permil (&) deviation from the “zero-delta” stan-
dard (IRMM 014, ERM-AE647, SRM 683)
! accuracy and precision of d56Fe, d65Cu, and d66Zn determination
56
ð56 Fe=54 FeÞsample
d Feð&Þ ¼ 56  1  1000 (2.1)
ð Fe=54 FeÞIRMM014
! analysis were evaluated in this study. To evaluate the matrix
65
ð65 Cu=63 CuÞsample
d Cuð&Þ ¼  1  1000 (2.2)
ð65 Cu=63 CuÞERM-AE647
!
ð66 Zn=64 ZnÞsample
66
d Znð&Þ ¼  1  1000 (2.3)
ð66 Zn=64 ZnÞSRM683
The reported d values of Cu and Zn in this study were con-
verted values using the following equations:32,33
d65CuNIST976 = d65CuERM-AE647 + 0.20& (2.4)
d66ZnJMC-Lyon = d66ZnSRM683 + 0.12& (2.5)
3. Accuracy and precision
3.1 Effects of acid molarity and concentration mismatch
during Fe, Cu and Zn isotope analysis
The possible inuence of varied acid molarity of sample and
standard on isotope analysis was evaluated by changing the acid
molarity (1 wt% HNO3 ∼ 3 wt% HNO3) of samples (IRMM 014,
ERM 647, SRM 683 for Fe, Cu and Zn, respectively), and using
the certain acid molarity (2 wt% HNO3) of standards (IRMM
014, ERM 647, SRM 683 for Fe, Cu and Zn, respectively). The
possible inuence of concentration mismatch was evaluated by
changing the (Fe, Cu or Zn) concentrations of samples (Csample/
Cstandard = 0.5 ∼ 2), and using the certain metal concentration
of standards (2.5 mg g−1 for Fe, 200 ng g−1 for Cu and 100 ng g−1
for Zn). The results showed that there is no signicant effect on
d65Cu and d66Zn measurement results with HNO3 concentration
of samples varied from 1 wt% to 2.5 wt%, and Csample/Cstandard
varied from 0.5 to 2. When the acid molarity of sample was
increased to 3 wt%, the d65Cu analysis result is obviously lower
than the reference value d65Cu = 0 (Fig. 2).
During Fe isotope analysis, the mismatch of acid molarity
between samples and standards will result in a large shi in
d56Fe values. For example, a 25% difference of acid molarity
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between samples and standards caused a shi of d56Fe values by
larger than 0.08&. In order to eliminate the effect of acid
molarity, the same batch of 2% HNO3 was always used for
sample and standards dilution during isotope analysis in this
study. When the Csample/Cstandard varied from 0.8 to 2, there is no
signicant effect on d56Fe measurement. But when the Csample/
Cstandard is 0.5, the measured d56Fe value is 0.07& lower than
the reference value. The concentration of Fe, Cu and Zn in
samples is set within ±10% of the standards to eliminate the
effect of concentration mismatch.
3.2 Effects of matrix elements and isobaric interferences
during Fe, Cu and Zn isotope analysis
The matrix elements and isobaric interference could affect the
on MC-ICP-MS. The effects of matrix elements and the elements
which could cause isobaric interference on Fe and Zn isotope
effects on Fe isotope analysis, different proportions of major
elements (such as Na, Mg, Al, K, and Ca) were doped into IRMM
014 solution, and the mixed solutions have been analysed (Fig. 3
and S2†). The results exhibit that when Na/Fe $ 0.5, Mg/Fe $
0.5, K/Fe $ 0.5, and Ca/Fe $ 0.5, the matrix effects are signi-
cant that the measured d56Fe values obviously deviate from the
reference value (d56Fe = 0). The effects of isobaric interference
of Cr and Ni (54Cr+ on 54 and 58Ni+ on 58) on Fe isotopes are
different in this study (Fig. 3). When the Ni/Fe increases to 0.5,
the measured d56Fe is still same as the reference value. But
when Cr/Fe = 0.0005, it can yield d56Fe value that deviate from
the reference value by −0.16&. The inuence of Cr on Fe
isotope analysis toward a light isotope composition is likely
a result of 54Cr+ interference on Fe (54Fe+). Therefore, the
monitoring of Cr is very important in the measurement of Fe
isotopes, and the ratio of Cr/Fe of sample solution should be
strictly lower than 0.0001.
The effects of matrix elements and polyatomic interferences
on Cu isotope analysis of the same instrument have been
evaluated in previous study.34 The measured d65Cu values
obviously deviate from the reference value when Na/Cu $ 1, Mg/
Cu $ 5, Al/Cu $ 3, and Fe/Cu $ 5. Cu isotope analysis is
sensitive to the presence of Ti, which could produce polyatomic
interferences on Cu masses, following their combination with O
and/or H (16O47Ti, 16O49Ti, 16O1H46Ti and 16O1H48Ti) on mass
63 and 65. Therefore, when Ti/Cu $ 0.2, it would signicantly
affect Cu isotope analysis.
The effects of matrix elements (Na, K and Ca), isobaric
interference (Ni), elements could produce potential polyatomic
interferences (e.g., Mg, Al, Cu and Ti), and doubly charged
species (Cd and Ba) on Zn isotope analysis were evaluated in
this study (Fig. 3 and S2†). The results show that Zn isotope
determinations have different tolerance with different
elements. The addition of Na, K, Ca, Cu, and Cd has negligible
effect on d66Zn values, when Na/Zn # 5, K/Zn # 5, Ca/Zn # 5,
Cu/Zn # 5 and Cd/Zn # 5. The d66Zn values are sensitive to the
presence of Mg, Al, Ni, Ti and Ba, which may be attributed to the
ion intense isobaric interferences on 64Zn+ produced by
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Fig. 2 The effects of different HNO3 acid strengths and concentration mismatches between samples and standard on d56Fe (a, b), d65Cu (c, d)
and d66Zn (e, f) analyses. The error bars represent the difference between two replicate measurements.

24
Mg40Ar+, 64Ni+ and 16O48Ti+, 67Zn+ produced by 27Al40Ar+. And
134
Ba and 136Ba can interfere as doubly charged species on 67Zn
and 68Zn, respectively. The measured d66Zn values obviously
deviate from the reference value when Mg/Zn $ 0.05, Al/Zn $
0.5, Ni/Zn $ 0.005, Ti/Zn $ 0.01, and Ba/Zn $ 0.1.
To overcome the effects from matrix elements, it is necessary
to eliminate these elements from sample cut during purica-
tion. Most major elements such as Na, Mg, Al, K and Ca were
removed by 6 mL 6 mol L−1 HCl and the ratios of major
elements to Fe, Cu and Zn were less than 0.01 aer one column

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Fig. 3 Doping experiments to test the isobaric interference and potential polyatomic interferences on d56Fe (a, b) and d66Zn (c–g) analysis. The
Fe and Zn concentrations for samples and bracketing standards (IRMM 014 for Fe and SRM 683 for Zn) are same.

purication. The low signal of matrix elements yielded neglec- amounts of Cr and Ni, and the Zn cut contains some Cd, which
ted inuence on isotope measurement. However, the collected are isobaric or molecular interferences and might affect Cu, Fe
Cu cut contains a small amount of Ti, the Fe cut contains small and Zn isotope analysis (Fig. 3, S1 and S2†). To avoid the

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interference effects, all of the puried Fe, Cu and Zn cuts of
biological reference materials in this study were measured by
ICP-MS (ELAN DRC II ICP-MS, PE, Thermo Fisher Scientic) to
ensure that their Ti/Cu < 0.1, Cr/Fe < 0.0001, Ni/Fe < 0.5, and Cd/
Zn < 5. If any sample solution had element/Fe (Cu or Zn) ratios
higher than these ratios, the second column would be pro-
cessed to eliminate the potential interference effect. The
isotopes were only analysed when the interference amounts
were lower than the range listed above.
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3.3 Double-spike calibration for Zn isotope analysis
The double-spike (DS) method was used to correct instrumental
mass bias produced by mass spectrometry for Zn isotope anal-
ysis. The double spike was composed of the mono-isotopic 67Zn
and 70Zn spikes obtained from Isoex USA (San Francisco, CA,
USA). The suitable isotope composition for 70Zn–67Zn DS is
accordance with the optimal ORNL DS composition outlined by
Rudge et al.,35 where 70Zn/67Zn = 1.08. Following mixing and
equilibration, the optimal spike/sample is evaluated by
changing spike concentrations at certain sample concentration,
with a range of spike/sample ratios from 0.2 to 5 (Fig. 4). When
the spike/sample ratio is in the range of 0.5 and 5, the measured
d66Zn values are same as the reference value, indicating that Zn
isotopes could be accurately and precisely analysed within this
spike/sample range.
3.4 Precision and accuracy
The long-term precision of isotope analysis was monitored by
in-house standard solutions. The average d56Fe values of the two
pure Fe solutions GSB and USTC-Fe were 0.73 ± 0.04& (2SD, n
= 41) and 0.70 ± 0.04& (2SD, n = 70), respectively. The average
d65Cu values of NIST SRM-976 and AAS (Cu) were 0.00 ± 0.04&
(2SD, n = 71) and 0.31 ± 0.04& (2SD, n = 51), respectively. The
average d66Zn values of AAS (Zn) and IRMM 3702 Zn solutions
were 0.04 ± 0.04& (2SD, n = 67) and 0.27 ± 0.04& (2SD, n =
42), respectively. n is the number of repeated measurements of
Fig. 4 d66Zn variations of SRM 683 standard solutions spiked with
different DS proportion.
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the same solution, while N is the number of independent
analyses of the same sample. The isotope data of six pure
solutions analysed over the last 12 months are shown in
Fig. S3,† and the precision of long-term analysis for each one is
better than 0.04& (2SD).
In this study, three ways are used to evaluate the precision and
accuracy of Fe, Cu and Zn isotope analysis of biological reference
materials. First, we made a synthetic solution including different
proportions of USTC-Fe, ERM-AE647 (Cu), and SRM 683 (Zn) with
known isotope compositions, and matrix elements such as Na,
Mg, Al, K, Ca, Ti, Cr, Mn, Ni and Cd to simulate biological
samples. Aer purication, the d56Fe value of USTC-Fe was
0.67 ± 0.02& (2SD, n = 3), which was consistent with the re-
ported value of 0.698 ± 0.05& (2SD, n = 1424).38 The d65Cu value
of ERM-AE647 (Cu) was −0.21 ± 0.03& (2SD, n = 3), which
was in agreement with the reported data of −0.21 ± 0.05&
(2SD, n = 60) within uncertainty.39 The average d66Zn value of
SRM 683 (Zn) was −0.12 ± 0.00& (2SD, n = 2), which was
consistent with the reported value of −0.12 ± 0.04& (2SD,
n = 18).36 This indicates there is no isotope fractionation during
chemical purication and instrumental measurements.
Second, different aliquots of the same sample powders were
independently digested and their isotopes were analysed to
ensure the precision and accuracy of isotope analysis. The
isotope data are listed in Table 2. Previous studies showed that
the Zn isotope composition of ERM-BB186 was heterogeneous,25
so ERM-BB186 was independently digested seven times and one
of digested ERM-BB186 aliquot was divided into two solutions (E-
1 and E-2) for separate purication to evaluate its homogeneity.
In this study, the d66Zn values of ERM-BB186 are variable
(−0.47& to −0.26&), which is consistent with previously re-
ported data.25,40,41 The d66Zn values of E-1 and E-2 are −0.45 ±
0.01& and −0.44 ± 0.02&, respectively, which are consistent
within the uncertain range. This indicated the same solution had
homogeneous Zn isotopes, but the different aliquots of ERM-
BB186 powder had heterogeneous Zn isotopes. Therefore, ERM-
BB186 is not suitable as a reference material for Zn isotopes. In
this study, GBW(E)0910933 was taken from different batches of
serum samples, which is reasonable that they have different
isotope compositions. Except for Zn isotopes of ERM-BB186 and
Fe isotopes and Cu isotopes of GBW(E)0910933, the isotope
values of replicated samples of all reference materials were the
same within the range of analytical uncertainty (Table 2).
Third, the Fe, Cu and Zn isotopes of ERM-BB186, which have
been reported in previous studies (Table S1†), were measured to
verify the accuracy of this analysis method. The d56Fe and d65Cu
values of ERM-BB186 in this study are −2.07 ± 0.05& (2SD, N =
8), and 2.76 ± 0.06& (2SD, N = 7), respectively. These are
consistent with the values reported by Rodushkin et al.40 (d56Fe =
−2.07 ± 0.14& (2SD, N = 8), d65Cu = 2.70 ± 0.25& (2SD, N = 8))
and Kubik et al.5 (d56Fe = −2.16 ± 0.09& (2SD, n = 6)) within
uncertainty. As we discussed above, Zn isotopes are heteroge-
neous in ERM-BB186. The d66Zn values reported in this study
varied from −0.26& to −0.47&, with an average value of −0.43 ±
0.14& (2SD, N = 8). The d66Zn value reported by Rodushkin et al.40
is −0.33 ± 0.11& (2SD, N = 8), by Schilling et al.41 is −0.41 ±
0.14& (2SD, N = 12), and by Moore et al.25 is −0.37 ± 0.11& (2SD,
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Table 2 Fe, Cu and Zn isotope compositions of nine biological reference materials reported in this studya

Fe Cu Zn
Sample Description (mg g−1) d56Fe (&) 2SD n (mg g−1) d65Cu (&) 2SD n (mg g−1) d66Zn (&) 2SD n

GBW10054 Skimmed soybean −0.53 0.05 3 0.52 0.05 3 0.85 0.07 2

−0.43 0.03 3 0.56 0.01 3 0.85 0.01 2
−0.47 0.05 3 0.52 0.03 3 0.89 0.02 2
Average (N = 3) 154 −0.48 0.10 9 15 0.53 0.05 9 45 0.86 0.05 6
GBW10055 Whole soybean −1.26 0.07 3 0.43 0.04 3 0.89 0.01 2
−1.23 0.02 3 0.43 0.02 3 0.87 0.01 2
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−1.22 0.01 3 0.44 0.04 3 0.87 0.03 2

−1.19 0.04 3 0.45 0.01 3 0.85 0.02 2
Average (N = 4) 53 −1.23 0.06 12 10 0.44 0.02 12 36 0.87 0.03 8
GBW(E)100199 Pumpkin −0.22 0.03 3 0.43 0.02 3 0.63 0.00 2
−0.19 0.04 3 0.42 0.02 3 0.63 0.00 2
−0.22 0.04 3 0.42 0.07 6 0.61 0.02 2
Average (N = 3) 41 −0.21 0.03 9 3 0.43 0.01 12 12 0.62 0.02 6
GBW10050 Prawn 0.12 0.02 3 0.41 0.01 3 0.29 0.04 2
0.13 0.02 3 0.39 0.03 3 0.29 0.02 2
0.10 0.03 3 0.37 0.02 3 0.28 0.02 2
0.11 0.03 3 0.40 0.01 3 0.26 0.03 2
Average (N = 4) 112 0.11 0.03 12 10 0.39 0.03 12 76 0.28 0.03 8
GBW09101b Human hair 0.05 0.03 3 −0.11 0.03 3 −0.15 0.07 4
0.07 0.01 3 −0.14 0.01 3 −0.14 0.02 2
0.05 0.05 3 −0.17 0.03 3 −0.14 0.02 2
0.06 0.04 3 −0.19 0.01 3 −0.13 0.01 2
Average (N = 4) 160 0.06 0.02 9 34 −0.15 0.07 12 191 −0.14 0.02 10
GBW(E)0910933 Human serum −1.92 0.04 2 0.14 0.03 3 −0.19 0.00 2
−1.84 0.03 2 0.25 0.01 3 −0.16 0.03 2
Average (N = 2) 5 −1.88 0.11 4 3 0.19 0.15 6 3 −0.17 0.04 4
GBW(E)100198 Yolk −2.41 0.04 3 −0.33 0.01 3 0.24 0.01 2
−2.38 0.03 3 −0.32 0.02 3 0.24 0.00 2
−2.42 0.02 3 −0.33 0.02 3 0.24 0.01 2
Average (N = 3) 123 −2.40 0.04 9 3 −0.33 0.01 9 76 0.24 0.01 6
GBW10051 Pork liver −1.74 0.04 3 0.41 0.02 3 −0.65 0.02 2
−1.76 0.05 3 0.42 0.01 3 −0.65 0.02 2
−1.75 0.04 3 0.46 0.02 3 −0.66 0.01 2
Average (N = 3) 519 −1.75 0.02 9 52 0.43 0.05 9 211 −0.65 0.02 6
ERM-BB186 Pig kidney −2.04 0.05 3 2.74 0.04 3 −0.26 0.01 5
−2.07 0.05 3 2.77 0.02 3 −0.47 0.01 2
−2.04 0.05 3 2.75 0.02 3 −0.46 0.03 2
−2.05 0.05 3 2.82 0.02 3 −0.45 0.04 2
−2.09 0.07 3 2.75 0.02 3 −0.45 0.00 2
−2.09 0.06 3 2.78 0.01 3 −0.44 0.03 2
E-1 −2.10 0.05 3 2.73 0.02 3 −0.45 0.01 2
E-2 −2.08 0.07 3 −0.44 0.02 2
Average (N = 8) 255 −2.07 0.05 24 36 2.76 0.06 21 134 −0.43 0.14 19
a
n is the number of repeated measurements of the same solution. N is the number of independent analyzes of the same sample. 2SD is two times
the standard deviation of n repeated measurements of the reference materials. The metal contents of samples (GBW10054, GBW10055, GBW(E)
100199, GBW10050, GBW09101b, GBW(E)0910933, GBW(E)100198 and GBW10051) were from https://www.ncrm.org.cn/. The metal contents of
ERM-BB186 were from https://ec.europa.eu/jrc/.

N = 8). Therefore, the d66Zn values of this study are in the same
variation range as previous studies (−0.45& to −0.27&).25,40,41 All
of the above support that the analysis method used in this study is
robust, and can provide precise and accurate isotope data.
4. Fe, Cu and Zn isotope ratios of
biological reference materials
56 65 66
The d Fe, d Cu and d Zn values of biological samples reported
in this study and previous studies are shown in Fig. 5 and 6. The
dataset can be divided into two groups, one for plants
(GBW10054, GBW10055, GBW(E)100199) and a marine animal
(GBW10050), and the other for human organs (GBW09101b,
GBW(E)0910933) and terrestrial animal organs (GBW(E)100198,
ERM-BB186, GBW10051), which show different Cu, Fe and Zn
isotope compositions. The total variations of d56Fe, d65Cu and
d66Zn values are 2.51&, 3.09& and 1.52&, respectively.
4.1 Fe isotope compositions
In Fig. 5, most plants and marine animals (−1.23& ∼ 0.11&)
have higher d56Fe values than terrestrial animals and human
organs (−2.40& ∼ 0.06&), which is consistent with the results

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Fig. 5 d56Fe, d65Cu and d66Zn values for biological reference materials
analysed in this study. (a) d66Zn versus d56Fe, (b) d56Fe versus d65Cu, and
(c) d66Zn versus d65Cu of nine biological reference materials. The
published date is shown with a gray pentagram (Rodushkin et al.40).
The error bars represent two times the standard deviation (2SD) of the
population of n repeat measurement (n $ 3) or the difference between
two replicate measurements (n < 3).
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JAAS
of Kubik et al.5 However, both whole soybean and skimmed
soybean present lighter Fe isotope compositions than other
plants, especially whole soybean, whose d56Fe value (−1.23 ±
0.06&, 2SD, N = 4) is even close to pig liver with d56Fe = −1.75
± 0.02& (2SD, N = 3). This might be related to the mechanism
of soybeans absorbing Fe. When soybeans absorb Fe from the
soil, they release protons into the soil to reduce the soil pH to
activate the immobile Fe3+. Then, the Fe3+ is reduced to Fe2+ in
the root, and absorbed by soybeans. Fe2+ is enriched in the
lighter isotopes due to its stronger bond strength than Fe3+, so
this process leads to the enrichment of light Fe isotopes in
soybeans.
Fe isotopes can be tracers of various metabolic pathways and
have been used to trace many diseases related to Fe metabo-
lism, such as hemochromatosis and ACD.12 The Fe isotope
compositions of biological reference materials are relatively
rare, especially human organs, because of the scarcity of human
materials. It is necessary to nd reference materials similar to
human organs, which could provide references in the future
study of human diseases. The reference materials from human
organs present different d56Fe values with −1.88 ± 0.11& (2SD,
N = 2) for human serum and 0.09 ± 0.08& (2SD, N = 4) for
human hair. Human hair presents higher values than other
terrestrial animal organs. The Fe isotope compositions of
human hair tested in this study (GBW09101b) are heavier than
ERM DB001 (d56Fe = −0.35& ± 0.24&, 2SD, n = 6) measured by
Kubik et al.5 (Fig. 6), but much lighter than human hair samples
(d56Fe range from −3.6& to −3.3&) tested by Walczyk and von
Blanckenburg.42 This indicates that Fe isotope compositions for
human hair are highly variable, which may be caused by envi-
ronmental variation.
Compared with plants and marine animals, the Fe isotope
compositions of human serum are more similar to those of
terrestrial animal organs, with light Fe isotope compositions
(Fig. 5 and 6). Therefore, terrestrial animal organs are very
suitable for preliminary investigation and medical research.
Pork liver (GBW10051) and pig kidney (ERM-BB186) present
different d56Fe values of −1.75 ± 0.02& (2SD, N = 3) and −2.07
± 0.05& (2SD, N = 8), respectively, which is controlled by the
different processes they absorb Fe. In most mammals, Fe is
absorbed by the intestine, where dietary Fe (II or III) is reduced
to Fe (II) for cellular needs and light Fe isotopes are preferen-
tially absorbed by cells (including cells in the kidney). Excess Fe
(II) with light Fe isotopes is exported back to the blood stream or
re-oxidized to Fe (III) enriched in heavy Fe isotopes and stored
within the liver as ferritin.43 Therefore, it could be concluded
that healthy human and animal's liver have much higher d56Fe
values than those of kidney and serum. This can provide
information for the study of diseases related to the Fe absorp-
tion process in the body.
Hemochromatosis is caused by the disrupted function of
hepcidin and ferroportin, which control intestinal Fe absorp-
tion, and lead to ineffective absorption of Fe in the intestine and
Fe overload in tissues. Patients with hemochromatosis have
abnormal Fe isotopes, whose d56Fe values in blood are 0.2& ∼
0.4& heavier than those in healthy individuals. ACD patients
show the opposite results. The uptake of Fe is strongly down-
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Fig. 6 d56Fe, d65Cu and d66Zn values for biological reference materials reported in previous studies5,23,32,37,40,41,48,52–67 and in this study (blue
circles). Among published samples, some samples are sold out, which are represented by red hollow squares. Some samples are still available
for purchase, which are represented by blue hollow squares. The yellow dotted boxes represent available for purchase and have measured all
three isotopes.

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regulated with Fe isotope compositions approximately 0.5&
lighter than those of able-bodied persons.12 Therefore, Fe
isotopes can help us diagnose diseases. To further use Fe
isotopes to study the diseases related to Fe metabolism in the
body, it is necessary to analyse Fe isotopes of different organs of
terrestrial animals with similar matrices as human to provide
more useful information.
4.2 Cu isotope compositions
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GBW(E)0910933, GBW10051 and ERM-BB186). These are within
the variation ranges of Zn isotopes of different trophic levels in
previous studies (Fig. 6). The Zn isotope fractionation in plants
and animals is controlled by absorption processes.47 The d66Zn
values are relatively high in plant, herbivore or low-trophic
omnivore organs, ranging from −0.43& to 1.17&. Moderate
isotope compositions occur in omnivores such as cattle, pigs,
and humans, with d66Zn values varying from −0.65& to 0.53&,
while the d66Zn values vary from −1.02& to −0.34& in the

organs of carnivores, especially in large carnivores, such as

The Cu isotope compositions of four reference materials
dogsh, which are rich in relatively light Zn isotopes (Fig. 6).
sampled from plants and marine animal organs are relatively
These ndings have been widely used to determine the trophic
homogenous with d65Cu values ranging from 0.39& to 0.53&
level of diverse and extinct animals, and our reference can
(Fig. 5), while human organs and terrestrial animal organs
provide support.
presented more fractioned Cu isotope compositions with d65Cu
As a tracer, Zn isotopes are not only used to determine the
values from −0.33& to 2.76&. Moreover, in human and
trophic level,6 but also to diagnose human disease.22,24,48 So far,
terrestrial animals, there are large Cu isotope fractionations
the Zn isotope data of human tissues and organs are limited
between different organs. The d65Cu values of human hair and
due to the ethical issues. The d66Zn values of the human serum
human serum are −0.15 ± 0.07& (2SD, N = 4) and 0.19 ±
and hair analysed in this study were −0.17 ± 0.04& (2SD, N = 2)
0.15& (2SD, N = 2), respectively. Pig kidney present obviously
and −0.14 ± 0.02& (2SD, N = 4), respectively (Fig. 5), which are
higher d65Cu values than pork liver, which are 2.76 ± 0.06&
different from the human serum reference material (BCR-639)
(2SD, N = 7) and 0.43 ± 0.05& (2SD, N = 3), respectively.
in Moore et al.25 and Larner et al.23 (Fig. 6). BCR-639 has
Abnormally high d65Cu values of pig kidney were also observed
abnormally low d66Zn values, due to the addition of puried
in sheep kidney44 and mouse kidney,45 which may be related to
zinc solution to natural serum. This articial processing may
ascorbic acid degradation and oxalic acid excretion in terrestrial
limit the applicability of BCR-639. GBW(E)0910933 is derived
animal bodies.46 Multiple processes can fractionate Cu isotopes
from natural sources, and can be widely used. Moreover, it's
in human and terrestrial animals. Therefore, targeted reference
necessary to nd other reference materials with Zn isotopes and
materials are necessary to study the homeostasis of different
matrix compositions similar to human. Since the isotope
organs in animals.
compositions of Zn are related to the trophic level, it can be
Cu is a tumor promoter, and Cu isotopes are considered to
considered to look for omnivores similar to humans, such as
be an advanced indicator of cancer.17,19 Cancer cells exhibited
cattle and pigs.
enhanced glycolysis followed by generating lactate. Lactate
Recent studies found that combined Fe, Cu and Zn isotopes
chelates Cu2+ with heavier Cu isotopes than pyruvate, which is
of brain tissues of patients could provide very important infor-
the product of normal cell glycolysis. This lactate chelation
mation to study the pathogenesis of Alzheimer's disease (AD),
increases rapidly with increasing lactate concentrations.19
especially amyloid-beta (Ab) plaque precipitation and hyper-
Therefore, cancer cells tend to enrich relatively heavy Cu
phosphorylated tau accumulation.24 The accumulation of Ab
isotopes. Free Cu+ with light isotope compositions escapes from
plaques and hyperphosphorylated tau proteins changes the
lactate-rich cancer cells and is exported to serum, resulting in
bonding environment in the brains of AD patients, which can
light Cu isotopes in the serum of patients.20 Télouk et al.19 found
cause metal isotope fractionation.49 Moynier et al.50 found that
that compared with controls, the d65Cu values of serum in
compared with controls, the brains of patients with Alzheimer's
breast cancer patients are signicantly lower. They found that
disease were enriched in light Cu isotopes (D65CuAD-control =
the alarm threshold of d65Cu is −0.35& in the serum, and
−0.25&) and heavy Zn isotopes (D66ZnAD-control = 0.2&).
patients may die from breast cancer when the d65Cu value is
Lighter Fe isotopes are also observed in the brain and serum of
below that. Moreover, the turnover time of Cu in the human
mice with Alzheimer's disease.24 Considering the synergistic
body is very short (less than 20 days),45 but the turnover time of
effect of these three elements in promoting Alzheimer's disease,
molecular biomarkers such as CEA (carcinoembryonic antigen)
the combination of Fe, Cu and Zn isotopes may provide more
and CA15.3 (carcinoembryonic antigen 15.3) are much longer
comprehensive information. In addition, the isotope composi-
(several months).20 Therefore, the decline of d65Cu values in
tions of the brain and serum were correlated,51 so the isotope
patient serum is much quicker than the other biomarkers.
composition of serum standard material analysed in this study
can be a quality control in further exploring Alzheimer's
4.3 Zn isotope compositions disease.
The Zn isotope compositions are related to the trophic levels.
Fig. 5 shows that the d66Zn values decrease from low trophic 5. Conclusions
levels to high trophic levels. The d66Zn ranges of plants are
0.62& ∼ 0.87&, those of low trophic omnivores (GBW10050 In this study, a high-precision Fe–Cu–Zn isotope analysis
and GBW(E)100198) are 0.24& ∼ 0.28&, and those of high method for biological samples was developed using one-
trophic omnivores are −0.65& to −0.14& (GBW09101b, column separation procedure. The method can provide more

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JAAS
comprehensive Fe, Cu and Zn isotope information of the same
sample aliquot. Aer puried, the Fe, Cu and Zn isotope
compositions were measured by MC-ICP-MS. Several important
parameters such as matrix effects (Ti, Cr and Ni), mismatch in
acid molarities and concentration between samples and
bracketing standards and DS dosages were evaluated to ensure
the high precision and accuracy of isotope measurement. Based
on optimal parameters, the long-term external reproducibility is
better than 0.04& for the three metal isotopes. The Fe, Cu and
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Zn isotope compositions of nine biological reference materials
were reported, among which ERM-BB186 has been analysed in
previous studies. These reference materials can be divided into
two groups, one from plants and marine animals with higher
d56Fe values (−1.23& ∼ 0.11&), d66Zn values (0.28& ∼ 0.87&)
and relatively homogenous d65Cu values (0.39& ∼ 0.53&),
while the other from terrestrial animals and humans has lower
d56Fe values (−2.40& to 0.06&), d66Zn values (−0.65& to
0.24&) and relatively homogenous d65Cu values (−0.33& to
2.76&). The isotope measurements for biological standard
materials can also improve the application of Fe, Cu and Zn
isotopes in environmental geochemistry, paleontology,
biomedicine, and disease diagnosis.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work is nancially supported by Strategic Priority Research
Program of Chinese Academy of Sciences (XDB 41000000), the
National Natural Science Foundation of China (42173003), and
the West Light Foundation of the Chinese Academy of Sciences
(xbzg-zdsys-202108).
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