High Precision Zinc Stable Isotope Measurement of Certified Biological Reference Materials Using the Double Spike Technique and Multiple Collector-ICP-MS
Anal Bioanal Chem
High Precision Zinc Stable Isotope Measurement of Certified Biological Reference Materials Using the Double Spike Technique and Multiple Collector-ICP-MS
Rebekah E. T. Moore 0 1 2
Fiona Larner 0 1 2
Barry J. Coles 0 1 2
Mark Rehkämper 0 1 2
0 Department of Earth Sciences, University of Oxford , South Parks Road, Oxford OX1 3AN , UK
1 Department of Earth Science and Engineering, Imperial College London, Royal School of Mines , Prince Consort Rd, Kensington London SW7 2AZ , UK
2 Rebekah E. T. Moore
Biological reference materials with wellcharacterised stable isotope compositions are lacking in the field of 'isotope biochemistry', which seeks to understand bodily processes that rely on essential metals by determining metal stable isotope ratios. Here, we present Zn stable isotope data for six biological reference materials with certified trace metal concentrations: fish muscle, bovine muscle, pig kidney, human hair, human blood serum and human urine. Replicate analyses of multiple aliquots of each material achieved reproducibilities (2sd) of 0.04-0.13‰ for δ66/64Zn (which denotes the deviation of the 66Zn/64Zn ratio of a sample from a pure Zn reference material in parts per 1000). This implies only very minor isotopic heterogeneities within the samples, rendering them suitable as quality control materials for Zn isotope analyses. This endorsement is reinforced by (i) the close agreement of our Zn isotope data for two of the samples (bovine muscle and human blood serum) to previously published results for different batches of the same material and (ii) the similarity of the isotopic data for the samples (δ66/64Zn ≈ - 0.8 to 0.0‰) to previously published Zn isotope results for similar biological materials. Further tests revealed that the applied Zn separation procedure is sufficiently effective to enable accurate data acquisition even at low mass resolving power (M/ΔM ≈ 400), as measurements and analyses conducted at much higher mass resolution (M/ΔM ≈ 8500) delivered essentially identical results.
Stable isotopes; Zinc; Biological reference materials; Double spike; Multiple collector inductively coupled plasma mass spectrometry; Ion exchange chromatography
Zinc is an essential biological trace metal as a constituent of
hundreds of enzymes with important functions in
neurotransmission and for maintaining healthy immune and reproductive
]. Disturbances in Zn balance have been linked
directly to a number of biological disorders. Tissue Zn mass
fractions, hereafter simply referred to as Zn ‘concentrations’,
are commonly measured to investigate where and how Zn
metabolism varies and whether new early diagnostic tools
may be developed to combat diseases associated with such
A more sensitive way to investigate the metabolism of
essential metals is to measure both the metal concentration
and stable isotope composition. Such analyses on animal
] and human [
] tissue are best performed using
multiple collector inductively coupled plasma mass
spectrometry (MC-ICP-MS) . With such instruments, it is possible
to determine isotope ratios with a precision that is at least an
order of magnitude better compared to quadrupole or single
collector ICP-MS. This enables the identification of smaller
differences in isotope compositions, which in turn can help to
identify even very small anomalies in metal metabolism.
Despite this, MC-ICP-MS is currently not routinely used in
bio-medical research. This may primarily reflect the
unfamiliarity of medical researchers with the instrumentation and
methods required for high precision isotope analyses and/or
the comparatively high cost of the required instruments. In
addition, the throughput of methods that are presently in use
is severely limited by the time-consuming manual sample
preparation, which is required for separation of the analyte
element at high yield and purity prior to isotopic analysis
]. The emergence of new techniques to automate and
accelerate sample processing , however, will help to
further establish the value of precise stable isotope measurements
for bio-medical investigations.
Laboratories that conduct such research would benefit from
the availability of biological materials with well-characterised
stable isotope compositions for quality control of analyses,
documentation of data quality, and to aid method development
and validation. Here, we present high precision Zn isotope
data for six commercially available biological reference
material (RM) which are certified for concentrations of Zn and
other elements [
]. The isotopic data were obtained in
multiple analyses over a 9-month period. For comparison, an
overview of stable Zn isotope compositions for biological
materials is also provided. As well as assessing the suitability
of these RMs as quality control samples for isotopic research,
this study also scrutinised various analytical aspects of current
techniques, including material digestion, metal isolation,
choice of double spike to sample ratio  and MC-ICP-MS
Materials and methods
Samples and reagents
Sample preparation was performed in ISO Class 6 metal-free
environments with Class 4 laminar flow hoods in the Imperial
College London MAGIC Laboratories. AnalaR grade 6 M
HCl and 15.4 M HNO3 purified in a Teflon still, >18 MΩ
cm H2O and 30–32% Romil H2O2 were used throughout.
Pre-cleaned Savillex Teflon vials were employed for sample
storage throughout all preparation and measurement stages, in
order to minimise Zn blanks.
The materials chosen for Zn isotope characterisation were
concentration certified, powdered and freeze-dried materials
from the Institute of Reference Materials and Measurement
(IRMM): ERM-DB001 (human hair), ERM-BB422 (fish
(pollock) muscle), ERM-BB184 (bovine muscle) and
ERMBB186 (pig kidney). Also analysed were two batches of
Sigma Aldrich BCR-639 (human blood serum), which were
stored at −40 °C, and a single batch of concentration certified,
freeze-dried Seronorm Trace Elements Urine L-1. The latter
RM is distributed in glass vials that contain the dried urine,
which is dissolved in 5 ml H2O to give certified element
The Zn isotope compositions of the samples were
determined and are reported relative to a solution of IRMM-3702
Zn, whilst London Zn and JMC Lyon Zn solutions were
analysed as secondary reference materials. All samples and
standard solutions were doped with a 64Zn–67Zn double spike
solution. This highly enriched tracer is characterised by
64Zn/67Zn ≈ 2.5 whilst all other Zn isotopes contribute only
about 3% to the total Zn budget [
The powdered IRMM RMs were sampled either four or five
times (for pig kidney ERM-BB186), to obtain individual
aliquots of between 40 and 60 mg or ∼200 mg. Four 250 μl
samples were taken from each batch of human blood serum
BCR-639. For the urine, after transferring two of the powder
aliquots into Teflon vials and dissolving in 5 ml H2O, five 2 ml
urine samples were used: two from each diluted aliquot and
one mixture of the remaining 1 ml of both. All digests were
weighed using a 0.01-mg sensitivity balance.
The samples were broken down by microwave digestion
using an Ethos EZ oven, fitted with SK-10 High Pressure
Rotor, in acid-cleaned 100 ml Teflon vessels. A blank was
included with each set of digestions. A 3:2 mixture of
15.4 M HNO3 and H2O2 was added to each sample to a total
of 7 ml for Urine L-1 and 8 ml for all other samples. After
12 h, allowing for partial digestion, the vessels were placed in
the microwave oven, which was ramped up to a temperature
of 210 °C and held there for 90 min. Following cooling, the
sample digests were transferred into Savillex Teflon vials and
evaporated to dryness. The hair samples were not fully
digested with this method, and an organic residue remained
after drying. Full digestion was subsequently achieved by the
addition of 15.4 M HNO3 and refluxing at 160 °C for 12 h.
Following evaporation, each digest was dissolved in 2 M HCl
to make stock solutions with ∼1 μg/ml Zn (based on the
certified concentrations) and aliquots with either ∼250 or
∼800 ng Zn were taken from each. An amount of double spike
solution was then added to each aliquot to yield a molar ratio
of tracer-derived to natural Zn (S/N) of 1 ± 0.05. The resulting
solutions were dried, redissolved in 6 M HCl, left at 130 °C
overnight to allow the sample and double spike to equilibrate,
and dried again. In addition to this, various London Zn
samples were prepared with S/N ratios ranging from ∼0.75 to
∼1.25, to assess the sensitivity of the double spike data
reduction to changes in spike to sample ratios. Double spiked
solutions of the pure Zn RMs IRMM-3702 Zn, London Zn and
JMC Lyon Zn with S/N ≈ 1 were prepared in a similar manner.
The double spiked samples were redissolved in 1 M HCl
and processed through an established anion exchange
chromatography procedure [
] to isolate Zn from
the sample matrix. In brief, in-house shrink-fit Teflon
columns with resin bed diameters of 3.5 mm were loaded
with 250 μl pre-cleaned BioRad AG MP1 100–200 mesh
resin. Before sample loading, the resin was cleaned with
2 ml 0.1 M HNO3 and 2 ml H2O, conditioned with 3 ml
6 M HCl and equilibrated using four aliquots of 0.5 ml
1 M HCl. The samples were loaded onto the resin in 1 ml
1 M HCl. Matrix elements were first removed using 8 ml
1 M HCl and the Zn was then eluted in 6 ml 0.01 M HCl
for collection in Teflon vials. After drying, 50 μl 15.4 M
HNO3 was twice added to each sample and evaporated to
dryness, to remove chloride ions and convert the samples
into nitrate form for isotope analysis. A London Zn
solution was also processed through the ion exchange
procedure with each batch of samples for quality control.
Zinc stable isotope compositions were determined at the
Imperial College London MAGIC Laboratory using a Nu
Plasma HR MC-ICP-MS and an Aridus II (CETAC
Technologies) sample introduction system fitted with glass
nebulisers that had typical solution uptake rates of ∼100 μl/
min. Faraday collectors L3, Ax, H2 and H4 were used to
measure the ion beams of 64Zn, 66Zn, 67Zn and 68Zn,
respectively, whilst 62Ni and 135Ba2+ (at mass 67.5) were monitored
on collectors L5 and H3 to enable corrections for spectral
interferences from isobaric Ni and doubly charged Ba ions
on Zn isotopes. All collectors were fitted with 1011 Ω resistors.
Each sample measurement consisted of three blocks of twenty
5 s integration cycles. Electronic baselines were measured for
15 s prior to each block whilst the ion beam was deflected in
the electrostatic analyser.
Both low and medium mass resolution were used to
measure the Zn isotope ratios of the RMs. Sample and reference
material solutions were prepared with 0.1 M HNO3 to Zn
concentrations of 100 ng/ml or 400 ng/g for measurements
at low and medium mass resolution, respectively. At low mass
resolution with M/ΔM ≈ 400 (where ΔM is the peak width
between 5 and 95% of full peak height), the sensitivity of the
instrument was typically ∼120 V/ppm, for a transmission
efficiency of ∼0.05%. A medium mass resolution with
M/ΔM ≈ 8500 was achieved by adjusting the source defining
slit. At such conditions, the sensitivity was typically ∼17 V/
ppm, which translates to a much lower transmission efficiency
The Zn isotope compositions are reported as δ66/64Zn,
expressed in parts per thousand (‰), which was calculated
by finding the relative difference between the 66Zn/64Zn ratios
of the sample and a standard (Eq. 1):
Here, IRMM-3702 Zn was used as the δ = 0 standard for all
reference material analyses and runs of IRMM-3702 Zn
bracketed all sample measurements to enable close
monitoring of any drift in instrumental mass bias. The standards
solutions that were used for the determination of δ66/64Zn values
thereby featured S/N values and Zn concentrations, which
matched the respective samples within 5 and 10%,
respectively. Following acquisition of the ‘raw’ isotopic data, all further
data reduction, including instrumental mass bias correction
via the double spike [
], corrections for the spectral
interferences from Ni+ and Ba2+ ions, and calculation of the final
δ66/64Zn values for samples, were carried out offline using an
iterative procedure [
]. For quality control, two samples
of London Zn were processed and analysed with each batch of
RMs, one with anion exchange chromatography and one
without, and their δ66/64Zn offsets from IRMM-3702 Zn were
monitored. The Zn concentrations were calculated offline
based on the isotope dilution technique using the 66Zn/68Zn
ratios determined for the sample-spike mixtures in the isotopic
runs, following correction for instrumental mass fractionation.
Results and discussion
Evaluation of sample preparation procedures
A number of tests were conducted to ascertain the reliability of
the sample preparation techniques used in this study for the
precise determination of stable Zn isotope compositions. Total
procedural Zn blanks ranged between 1 and 6 ng (n = 8). Such
amounts are essentially negligible, as even the maximum
blank would alter the δ66/64Zn value of a 250-ng Zn sample
by less than 0.02‰, assuming an isotopic offset of 1‰
between blank and sample.
The yield of the anion exchange separation protocol was
determined by comparing the expected and observed Zn ion
beam intensities during the isotopic measurements and found
to be >90% consistently for both the biological RMs and
London Zn solutions. In addition, analyses of London Zn
samples that were processed by anion exchange
chromatography yielded δ66/64Zn values that were identical to unprocessed
London Zn solutions (Table 1), thereby demonstrating that
accurate results are achieved even if the chemical yields are
The acquisition of accurate results is further supported by
the very low levels of spectral interferences from Ni and Ba
isotopes, as monitored during analyses of the biological
This study: unprocesseda
This study: processed
This study: all
Moeller et al. 2012 [
Archer et al. 2017 [
Moeller et al. 2012 [
Van Heghe et al. 2012 [
The 2sd and 2se values were calculated from repeat analyses of the RMs. The total number of measurements for
each RM and the number of individual sample aliquots that were processed through separation chemistry are
denoted by ‘n’ and ‘m’, respectively
a London Zn that was not ‘processed’ through the anion exchange chromatography procedure
reference samples. In detail, the measurements revealed a
typical contribution of 3 ppm 64Ni+ to the total ion beam at mass
64 and of <1, 5 and 20 ppm for Ba2+ ions at masses 66, 67 and
68, respectively. Given these low levels, the applied
interference corrections are negligible for all isotopes except 136Ba2+,
where the small correction provides accurate results that are
not subject to a systematic bias.
Results for pure Zn reference materials—London Zn and JMC Lyon Zn
Individual analyses of the Zn standard solutions consistently
produced ‘internal’ within-run precisions (2se) of about
±0.05‰ for δ66/64Zn. The external, between-run precision
(2sd), as determined from multiple analyses of both RMs,
was similar or only slightly worse at about ±0.05 to ±0.08‰
(Table 1). The mean δ66/64Zn values of London Zn and JMC
Lyon Zn, relative to IRMM-3702 Zn, were −0.20 ± 0.07 and
−0.32 ± 0.06‰, respectively. These results are fully consistent
with previous measurements of different aliquots of these
solutions (Table 1).
London Zn solutions that were spiked to obtain variable
S/N values from about 0.75 to 1.25 and analysed relative to
a London Zn solution with S/N ≈ 1 revealed no clearly
resolvable deviations from δ66/64Zn = 0.00 ± 0.06‰ (Fig. 1). The
data, however, suggest a possible trend of decreasing δ66/
64Zn with increasing S/N. This trend is less apparent if the
individual run results for the different S/N ratios are plotted
rather than the mean δ66/64Zn values obtained from the
replicate measurements, particularly because the analytical
precision is generally worse for samples with larger deviations
from S/N = 1 (see Electronic Supplementary Material,
ESM). Given these results, samples are best analysed relative
to δ = 0 standard solutions, which feature S/N ratios that
match to within about 15% or better (Fig. 1).
Results for biological reference materials
The Zn concentration and stable isotope data that were
obtained for the biological RMs are summarised in Table 2 and
Fig. 2, together with the certified Zn abundances and any
reference values from previous studies.
Overall, the measured Zn concentrations of the biological
RMs agree with the official certified values within the
combined 1sd uncertainties for all samples except the pig kidney
ERM-BB186 and the human urine (Trace Elements Urine
L-1) (Table 2). For the pig kidney, our analyses provide a
slightly higher Zn content of 144.5 ± 0.7 μg/g compared to
the certified concentration of 134 ± 5 μg/g. In the case of the
The 2sd data for the δ66/64 Zn values of individual digest are based on repeat analyses of the matching standard, whereas the 2sd and 2se for the mean δ66/
64 Zn values of each RM (italicised) are calculated from all individual RM measurements. The total number of measurements and the number of
individual sample aliquots that went through the separation chemistry for each digest and RM are denoted by ‘n’ and ‘m’, respectively
urine, we find a significant deviation from the certified Zn
concentration, with a result of 221 ± 8 ng/g versus a reference
value of 347 ± 70 ng/g. It is improbable that this difference is
primarily due to incomplete transfer of the sample powder
from the glass storage vials prior to initial sample dissolution
in 5 ml H2O (the volume at which the Zn concentration is
]). This is reinforced by the observation that
essentially identical Zn concentrations were measured for two
different glass vials of the urine RM even though the amount
of dry residue recovered from each vial differed slightly (at
136.36 and 126.43 mg). Hence, it is more likely that the low
Zn concentration determined here reflects either sample
heterogeneity amongst different aliquots or batches of the urine
RM or a high Zn blank during the original certification
For the isotopic analyses of the biological RMs, the internal
precision was similar to results obtained for pure Zn solutions
at about ±0.05‰ (2se) for δ66/64Zn. A similar result emerges
for the external reproducibility of data obtained for single
digests of the biological RMs that were analysed multiple
times and where some digests were split into two or three
aliquots for separate processing through the column chemistry
(Table 2). In detail, these measurements yielded precisions
(2sd) of ±0.04 to ±0.10‰, nearly identical to the
reproducibility seen for multiple London Zn and JMC Lyon Zn analyses
(Table 1). Together, these results demonstrate that the
separation procedure produces clean Zn cuts, which enable precise
isotopic analyses that are not affected by spectral or
The accuracy of the Zn stable isotope data can be
evaluated, as reference values from previous studies are available for
two of the materials, bovine muscle ERM-BB184 and human
blood serum BCR-639 (Table 2, Fig. 2). For the bovine
muscle, our δ66/64Zn result of −0.30 ± 0.10‰ agrees with data
from a different laboratory (−0.24 ± 0.02‰ [
BCR639, the average δ66/64Zn value found was −3.29 ± 0.12‰,
with no difference between the two batches (−3.31 ± 0.13
and 3.28 ± 0.11‰). The δ66/64Zn value of this sample is
identical to previous analyses of the RM, which were conducted
(unprocessed). For the human blood serum, the results are plotted on a
different y-axis, due to the large isotopic offset. The δ66/64Zn literature
data for London Zn, bovine muscle and human blood serum are from
Moeller et al. 2012 [
], Costas-Rodríguez et al. 2014 [
] and Larner
et al. 2015 [
by a different analyst on a separately purchased batch of the
]. Notably, these latter measurements yielded a
comparatively large uncertainty of ±0.2‰ (Table 2).
The δ66/64Zn data that were obtained for the remaining four
reference materials showed only limited variability, with
results of –0.34 ± 0.04‰ for fish muscle ERM-BB422, –0.65
± 0.13‰ for pig kidney ERM-BB186, –0.31 ± 0.04‰ for
human hair ERM-DB001 and −0.05 ± 0.04‰ for the Seronorm
Trace Elements Urine L-1 (Table 2, Fig. 2). Notably, the dried
residue in two glass vials of the urine RM produced solutions
with identical Zn isotope compositions. The latter result
suggests that the urine RM is likely to have a homogeneous Zn
isotope composition despite possible significant differences in
The isotopic analyses for four of the biological RMs were
carried out both at low and medium mass resolutions to better
ascertain the accuracy of the data. The results for the different
analysis modes are compared in Fig. 3. Importantly, this
diagram reveals no significant differences between these two sets
of data. This indicates that our sample preparation procedure
produces Zn solutions, which are sufficiently pure for accurate
δ66/64Zn measurements even at low mass resolution.
Importantly, this allows for more repeat analyses of individual
samples or smaller sample size, as much less Zn is needed for
a single measurement at low mass resolution than at medium
mass resolution, where ion beam transmission is much lower.
This may prove important in bio-medical studies because
tissue sample size is often limited and sampling is usually only
possible on a single occasion.
The overall mean values that were calculated for the six
biological RMs are based on at least eight individual analyses,
which were obtained on four to eight separate digests.
Notably, the 2sd uncertainties of the mean results vary
between ±0.04 and ±0.13‰ (Table 2). In detail, three of the
biological RMs reveal overall mean results with uncertainties
of ≥0.10‰ and which are hence slightly but noticeably larger
than the typical 2sd reproducibilities for multiple
measurements of a single digest (generally ≤0.08‰). It is possible that
this small discrepancy reflects minor sample heterogeneities,
which are just noticeable when sampling occurs at the 50 to
200 mg scale. However, given the current uncertainty of stable
Zn isotope analyses of about ±0.05 to ±0.15‰ (2sd), this
slight heterogeneity will have only a minor impact on any
Also of note is the observation that all but one of the
biological RMs have δ66/64Zn values of between about ±0.0 and –
0.8‰ and which hence fall within the range of stable isotope
compositions that were previously measured for such tissues
(Fig. 4). As such, they are particularly well-suited as quality
control materials for Zn isotope analyses. The exception is
BCR-639, which is about 3‰ lower in δ66/64Zn compared to
previous results obtained for human blood and blood serum
(Fig. 4). This large discrepancy is most likely related to the
preparation of this RM, which involved the addition of a Zn
solution (NIST SRM 3168a) to natural blood serum [
]. It is
also conceivable that this method of preparation may be
responsible for the possible minor isotopic heterogeneity of this
sample, as is indicted by the relatively poor reproducibility of
our overall mean result and the comparatively large difference
between the data obtained here and previously by Larner et al.
A double spike technique in conjunction with MC-ICP-MS
was used to measure the Zn isotope compositions of six
biological reference materials with certified trace metal
concentrations. It is shown that these methods are suitable for the
Fig. 4 Compilation of published δ66/64Zn values for animal tissue and
the reference materials analysed in this study (squares, except BCR-639),
reported relative to IRMM-3702 Zn [
16, 18, 21, 22, 25, 26, 29, 60–62
Literature data reported relative to JMC Lyon Zn was corrected for an
offset of 0.32‰ between IRMM-3702 Zn and JMC Lyon Zn (Table 1)
routine determination of δ66/64Zn with an external precision
(2sd) of about ±0.05 to ±0.10‰. Analyses of multiple digests
of the biological RMs yielded mean δ66/64Zn values with a
reproducibility of between ±0.04 and ±0.13‰. This suggests
that these RMs feature no or only very minor heterogeneities
in Zn isotope composition, when sampling occurs on the scale
of 50 to 200 mg. As such, all are in principle ideally suited as
quality control materials for Zn isotope measurements in
biomedical research. This conclusion is reinforced by the
observations that all samples, except for human blood serum
BCR639, also have Zn isotope compositions that fall within the
range of normal natural values. The unusually low δ66/64Zn
value of BCR-639 reflects that this sample was produced by
the addition of a purified Zn solution to natural blood and this
non-natural origin may limit the suitability of this material for
some applications. Further measurements revealed that
isotopic analyses of the samples at low and medium mass
resolution produced essentially identical Zn isotope data. This
demonstrates that the sample preparation procedure yields
sufficiently pure separates of Zn, such that the isotopic
measurements do not require medium mass resolution to resolve
remaining spectral interferences. This result is advantageous, as
low mass resolution analyses require significantly less Zn than
measurements at higher mass resolving power.
Acknowledgements This project was funded by a Janet Watson
Scholarship awarded to RETM and an STFC Impact Acceleration
Award to MR.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
Open Access This article is distributed under the terms of the Creative
C o m m o n s A t t r i b u t i o n 4 . 0 I n t e r n a t i o n a l L i c e n s e ( h t t p : / /
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
1. Bonaventura P , Benedetti G , Albarède F , Miossec P. Zinc and its role in immunity and inflammation . Autoimmun Rev . 2015 ; 14 : 277 - 85 . doi: 10 .1016/j.autrev. 2014 . 11 .008.
2. Tapiero H , Tew KD . Trace elements in human physiology and pathology: zinc and metallothioneins . Biomed Pharmacother . 2003 ; 57 : 399 - 411 . doi: 10 .1016/S0753- 3322 ( 03 ) 00081 - 7 .
3. Pluhator MM , Thomson ABR , Fedorak RN . Clinical aspects of trace elements: zinc in human nutrition-zinc deficiency and toxicity . Can J Gastroenterol . 1996 ; 10 : 97 - 103 .
4. Haase H , Rink L . Multiple impacts of zinc on immune function . Metallomics . 2014 ; 6 : 1175 . doi: 10 .1039/c3mt00353a.
5. Aranha LN , Lobo JC , Stockler-Pinto MB , Leal VDO , Torres JPM , Mafra D . Relationship between zinc levels and plasma leptin in hemodialysis patients . J Trace Elem Med Biol . 2012 ; 26 : 238 - 42 . doi: 10 .1016/j.jtemb. 2012 . 02 .007.
6. Panayi AE , Spyrou NM , Iversen BS , White MA , Part P. Determination of cadmium and zinc in Alzheimer's brain tissue using inductively coupled plasma mass spectrometry . J Neurol Sci . 2002 ; 195 : 1 - 10 . doi: 10 .1016/ S0022 -510X( 01 ) 00672 - 4 .
7. Zhao H-W , Lin J , Wang X -B, Cheng X , Wang J-Y , Hu B-L , et al. Assessing plasma levels of selenium, copper, iron and zinc in patients of parkinson's disease . PLoS One . 2013 ; 8:e83060 . doi: 10 . 1371/journal.pone. 0083060 .
8. Prasad AS . Impact of the discovery of human zinc deficiency on health . J Trace Elem Med Biol . 2014 ; 28 : 357 - 63 . doi: 10 .1016/j. jtemb. 2014 . 09 .002.
9. Kozlowski H , Luczkowski M , Remelli M , Valensin D . Copper, zinc and iron in neurodegenerative diseases (Alzheimer's, Parkinson's and prion diseases) . Coord Chem Rev . 2012 ; 256 : 2129 - 41 . doi: 10 . 1016/j.ccr. 2012 . 03 .013.
10. Bjorklund G. The role of zinc and copper in autism spectrum disorders . Acta Neurobiol Exp (Wars) . 2013 ; 73 : 225 - 36 .
11. Kawahara M , Mizuno D , Koyama H , Konoha K , Ohkawara S , Sadakane Y. Disruption of zinc homeostasis and the pathogenesis of senile dementia . Metallomics . 2014 ; 6 : 209 - 19 . doi: 10 .1039/ C3MT00257H.
12. Damphousse V , Mailhot M , Berthiaume Y , Rabasa-Lhoret R , Mailhot G. Plasma zinc in adults with cystic fibrosis: correlations with clinical outcomes . J Trace Elem Med Biol . 2014 ; 28 : 60 - 4 . doi: 10 .1016/j.jtemb. 2013 . 10 .003.
13. Stelmashook EV , Isaev NK , Genrikhs EE , Amelkina GA , Khaspekov LG , Skrebitsky VG , et al. Role of zinc and copper ions in the pathogenetic mechanisms of Alzheimer's and Parkinson's diseases . Biochem Biokhimiia . 2014 ; 79 : 391 - 6 . doi: 10 .1134/ S0006297914050022.
14. González-Domínguez R , García-Barrera T , Gómez-Ariza JL . Characterization of metal profiles in serum during the progression of Alzheimer's disease . Metallomics . 2014 ; 6 : 292 - 300 . doi: 10 . 1039/c3mt00301a.
15. Chiba M , Katayama K , Takeda R , Morita R , Iwahashi K , Onishi Y , et al. Diuretics aggravate zinc deficiency in patients with liver cirrhosis by increasing zinc excretion in urine . Hepatol Res . 2013 ; 43 : 365 - 73 . doi: 10 .1111/j.1872- 034X . 2012 . 01093 .x.
16. Balter V , Lamboux A , Zazzo A , Télouk P , Leverrier Y , Marvel J , et al. Contrasting Cu , Fe, and Zn isotopic patterns in organs and body fluids of mice and sheep, with emphasis on cellular fractionation . Metallomics . 2013 ; 5 : 1470 . doi: 10 .1039/c3mt00151b.
17. Balter V , Zazzo A , Moloney AP , Moynier F , Schmidt O , Monahan FJ , et al. Bodily variability of zinc natural isotope abundances in sheep . Rapid Commun Mass Spectrom . 2010 ; 24 : 605 - 12 . doi: 10 . 1002/rcm.4425.
18. Moynier F , Fujii T , Shaw AS , Le Borgne M. Heterogeneous distribution of natural zinc isotopes in mice . Metallomics . 2013 ; 5 : 693 . doi: 10 .1039/c3mt00008g.
19. Büchl A , Hawkesworth CJ , Ragnarsdottir KV , Brown DR . Repartitioning of Cu and Zn isotopes by modified protein expression . Geochem Trans . 2008 ; 9 : 11 . doi: 10 .1186/ 1467 -4866-9-11.
20. Hirata T , Tanoshima M , Suga A , Tanaka Y , Nagata Y , Shinohara A , et al. Isotopic analysis of calcium in blood plasma and bone from mouse samples by multiple collector-ICP-mass spectrometry . Anal Sci . 2008 ; 24 : 1501 - 7 .
21. Larner F , Woodley LN , Shousha S , Moyes A , Humphreys-Williams E , Strekopytov S , et al. Zinc isotopic compositions of breast cancer tissue . Metallomics . 2015 ; 7 : 112 - 7 . doi: 10 .1039/C4MT00260A.
22. Costas-Rodríguez M , Van Heghe L , Vanhaecke F . Evidence for a possible dietary effect on the isotopic composition of Zn in blood via isotopic analysis of food products by multi-collector ICP-mass spectrometry . Metallomics . 2014 ; 6 : 139 - 46 . doi: 10 .1039/ C3MT00244F.
23. Gulson B , Wong H , Korsch M , Gomez L , Casey P , McCall M , et al. Comparison of dermal absorption of zinc from different sunscreen formulations and differing UV exposure based on stable isotope tracing . Sci Total Environ . 2012 ; 420 : 313 - 8 . doi: 10 .1016/j. scitotenv. 2011 . 12 .046.
24. Jaouen K , Pons M-L , Balter V . Iron, copper and zinc isotopic fractionation up mammal trophic chains . Earth Planet Sci Lett . 2013 ; 374 : 164 - 72 . doi: 10 .1016/j.epsl. 2013 . 05 .037.
25. Van Heghe L , Deltombe O , Delanghe J , Depypere H , Vanhaecke F. The influence of menstrual blood loss and age on the isotopic composition of Cu, Fe and Zn in human whole blood . J Anal At Spectrom . 2014 ; 29 : 478 - 82 . doi: 10 .1039/C3JA50269D.
26. Van Heghe L , Engström E , Rodushkin I , Cloquet C , Vanhaecke F. Isotopic analysis of the metabolically relevant transition metals Cu, Fe and Zn in human blood from vegetarians and omnivores using multi-collector ICP-mass spectrometry . J Anal At Spectrom . 2012 ; 27 : 1327 . doi: 10 .1039/c2ja30070b.
27. Télouk P , Puisieux A , Fujii T , Balter V , Bondanese VP , Morel A-P , et al. Copper isotope effect in serum of cancer patients. A pilot study . Metallomics . 2015 ; 7 : 299 - 308 . doi: 10 .1039/C4MT00269E.
28. Costas-Rodríguez M , Anoshkina Y , Lauwens S , Van Vlierberghe H , Delanghe J , Vanhaecke F . Isotopic analysis of Cu in blood serum by multi-collector ICP-mass spectrometry: a new approach for the diagnosis and prognosis of liver cirrhosis? Metallomics . 2015 ; 7 : 491 - 8 . doi: 10 .1039/C4MT00319E.
29. Jaouen K , Gibert M , Lamboux A , Telouk P , Fourel F , Albarède F , et al. Is aging recorded in blood Cu and Zn isotope compositions? Metallomics . 2013 ; 5 : 1016 - 24 . doi: 10 .1039/C3MT00085K.
30. Larner F , Sampson B , Rehkämper M , Weiss DJ , Dainty JR , O'Riordan S , et al. High precision isotope measurements reveal poor control of copper metabolism in Parkinsonism . Metallomics. 2013 ; 5 : 125 . doi: 10 .1039/c3mt20238k.
31. Aramendía M , Rello L , Resano M , Vanhaecke F. Isotopic analysis of Cu in serum samples for diagnosis of Wilson's disease: a pilot study . J Anal At Spectrom . 2013 ; 28 : 675 . doi: 10 .1039/c3ja30349g.
32. Resano M , Aramendía M , Rello L , Calvo ML , Bérail S , Pécheyran C . Direct determination of Cu isotope ratios in dried urine spots by means of fs-LA-MC-ICPMS . Potential to diagnose Wilson's disease . J Anal At Spectrom . 2013 ; 28 : 98 - 106 . doi: 10 .1039/ C2JA30262D.
33. Balter V , Nogueira da Costa A , Bondanese VP , Jaouen K , Lamboux A , Sangrajrang S , et al. Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients . Proc Natl Acad Sci . 2015 ; 112 : 982 - 5 . doi: 10 .1073/pnas.1415151112.
34. Jaouen K , Balter V . Menopause effect on blood Fe and Cu isotope compositions . Am J Phys Anthropol . 2014 ; 153 : 280 - 5 . doi: 10 . 1002/ajpa.22430.
35. Jaouen K , Balter V , Herrscher E , Lamboux A , Telouk P , Albarède F. Fe and Cu stable isotopes in archeological human bones and their relationship to sex . Am J Phys Anthropol . 2012 ; 148 : 334 - 40 . doi: 10 .1002/ajpa.22053.
36. Albarède F , Telouk P , Lamboux A , Jaouen K , Balter V . Isotopic evidence of unaccounted for Fe and Cu erythropoietic pathways . Metallomics . 2011 ; 3 : 926 . doi: 10 .1039/c1mt00025j.
37. Anoshkina Y , Costas-Rodríguez M , Vanhaecke F. High-precision Fe isotopic analysis of whole blood for biomedical purposes without prior isolation of the target element . J Anal At Spectrom . 2015 ; 30 : 1816 - 21 . doi: 10 .1039/C5JA00209E.
38. von Blanckenburg F , Oelze M , Schmid DG , van Zuilen K , Gschwind H-P , Slade AJ , et al. An iron stable isotope comparison between human erythrocytes and plasma . Metallomics . 2014 ;6: 2052 - 61 . doi: 10 .1039/C4MT00124A.
39. Hotz K , Walczyk T . Natural iron isotopic composition of blood is an indicator of dietary iron absorption efficiency in humans . JBIC J Biol Inorg Chem . 2013 ; 18 : 1 - 7 . doi: 10 .1007/s00775-012-0943-7.
40. Van Heghe L , Delanghe J , Van Vlierberghe H , Vanhaecke F. The relationship between the iron isotopic composition of human whole blood and iron status parameters . Metallomics . 2013 ; 5 : 1503 . doi: 10 .1039/c3mt00054k.
41. von Blanckenburg F , Noordmann J , Guelke-Stelling M. The iron stable isotope fingerprint of the human diet . J Agric Food Chem . 2013 ; 61 : 11893 - 9 . doi: 10 .1021/jf402358n.
42. Hotz K , Krayenbuehl P-A , Walczyk T. Mobilization of storage iron is reflected in the iron isotopic composition of blood in humans . JBIC J Biol Inorg Chem . 2012 ; 17 : 301 - 9 . doi: 10 .1007/s00775-011- 0851-2.
43. Krayenbuehl P -A. Hereditary hemochromatosis is reflected in the iron isotope composition of blood . Blood . 2005 ; 105 : 3812 - 6 . doi: 10 .1182/blood-2004 -07-2807.
44. Morgan JLL , Skulan JL , Gordon GW , Romaniello SJ , Smith SM , Anbar AD . Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes . Proc Natl Acad Sci . 2012 ; 109 : 9989 - 94 . doi: 10 .1073/pnas.1119587109.
45. Rehkämper M , Schönbächler M , Stirling CH . Multiple collector ICP-MS: introduction to instrumentation, measurement techniques and analytical capabilities . Geostand Newsl . 2001 ; 25 : 23 - 40 . doi: 10 .1111/j. 1751 - 908X . 2001 .tb00785.x.
46. Archer C , Vance D . Mass discrimination correction in multiplecollector plasma source mass spectrometry: an example using Cu and Zn isotopes . J Anal At Spectrom . 2004 ; 19 : 656 . doi: 10 .1039/ b315853e.
47. Mason TFD , Weiss DJ , Horstwood M , Parrish RR , Russell SS , Mullane E , et al. High-precision Cu and Zn isotope analysis by plasma source mass spectrometry . J Anal At Spectrom . 2004 ; 19 : 209 . doi: 10 .1039/b306958c.
48. Institute for Reference Materials and Measurements (IRMM) (2005) Certificate of analysis ERM-BB184 . https://ec.europa.eu/ jrc/sites/default/files/rm/ERM-BB184_ cert .pdf
49. (IRMM) I for RM and M ( 2005 ) Certificate of analysis ERMBB186 . https://ec.europa.eu/jrc/sites/default/files/rm/ERMBB186_cert.pdf
50. Institute for Reference Materials and Measurements (IRMM) (2005) Certificate of analysis ERM-BB422 . http://www.erm-crm. org/ERM_products/search/certificates/BB422.pdf
51. Institute for Reference Materials and Measurements (IRMM) (2007) Certified Reference Material BCR- 639 . https://ec.europa. eu/jrc/sites/default/files/rm/BCR-639_cert.pdf
52. Institute for Reference Materials and Measurements (IRMM) (2013) Certificate of Analysis ERM-DB001 . https://ec.europa.eu/ jrc/sites/default/files/rm/ERM-DB001_ cert .pdf
53. Seronorm ( 2015 ) Seronorm TM Trace Elements Urine L-1 , Lot 1403080
54. Rudge JF , Reynolds BC , Bourdon B. The double spike toolbox . Chem Geol . 2009 ; 265 : 420 - 31 . doi: 10 .1016/j.chemgeo. 2009 . 05 . 010.
55. Arnold T , Schönbächler M , Rehkämper M , Dong S , Zhao F-J , Kirk GJD , et al. Measurement of zinc stable isotope ratios in biogeochemical matrices by double-spike MC-ICPMS and determination of the isotope ratio pool available for plants from soil . Anal Bioanal Chem . 2010 ; 398 : 3115 - 25 . doi: 10 .1007/s00216-010-4231-5.
56. Bridgestock LJ , Williams H , Rehkämper M , Larner F , Giscard MD , Hammond S , et al. Unlocking the zinc isotope systematics of iron meteorites . Earth Planet Sci Lett . 2014 ; 400 : 153 - 64 . doi: 10 .1016/j. epsl. 2014 . 05 .029.
57. Siebert C , Nägler TF , Kramers JD . Determination of molybdenum isotope fractionation by double-spike multicollector inductively coupled plasma mass spectrometry . Geochemistry Geophys Geosystems . 2001 ; 2 : 1032 - 16 . doi: 10 .1029/2000GC000124.
58. Moeller K , Schoenberg R , Pedersen R-B , Weiss D , Dong S. Calibration of the new certified reference materials ERM-AE633 and ERM-AE647 for copper and IRMM-3702 for zinc isotope amount ratio determinations . Geostand Geoanalytical Res . 2012 ; 36 : 177 - 99 . doi: 10 .1111/j. 1751 - 908X . 2011 . 00153 .x.
59. Archer C , Andersen MB , Cloquet C , Conway TM , Dong S , Ellwood M , et al. Inter-calibration of a proposed new primary reference standard AA-ETH Zn for zinc isotopic analysis . J Anal At Spectrom . 2017 ; 226 : 280 - 97 . doi: 10 .1039/C6JA00282J.
60. Stenberg A , Andrén H , Malinovsky D , Engström E , Rodushkin I , Baxter DC . Isotopic variations of Zn in biological materials . Anal Chem . 2004 ; 76 : 3971 - 8 . doi: 10 .1021/ac049698f.
61. Ohno T , Shinohara A , Chiba M , Hirata T. Precise Zn isotopic ratio measurements of human red blood cell and hair samples by multiple collector-ICP-mass spectrometry . Anal Sci . 2005 ; 21 : 425 - 8 . doi: 10 . 2116/analsci.21.425.
62. Maréchal CN , Télouk P , Albarède F . Precise analysis of copper and zinc isotopic compositions by plasma-source mass spectrometry . Chem Geol . 1999 ; 156 : 251 - 73 . doi: 10 .1016/S0009- 2541 ( 98 ) 00191 - 0 .
63. Byrialsen K , Kristiansen J , Christensen JM , Dirscherl C , Gawlik BM , Klein CL , Lamberty A ( 2004 ) The certification of the mass concentrations of aluminium, selenium and zinc in human serum (BCR-637 , BCR-638, BCR- 639 ). Inst. Ref. Mater. Meas.