Clinical Chemistry 46: 1781-1786, 2000;
(Clinical Chemistry. 2000;46:1781-1786.)
© 2000 American Association for Clinical Chemistry, Inc.
Human Urine Certified Reference Material for Arsenic Speciation
Jun Yoshinaga1,a,
Amit Chatterjee1,
Yasuyuki Shibata1,
Masatoshi Morita1 and
John S. Edmonds2
1
National Institute for Environmental Studies (NIES), 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan.
2
Department of Chemistry, De Montfort University, The
Gateway, Leicester LE1 9BH, United Kingdom.
a Address correspondence to this author at: Institute of Environmental Studies, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. Fax 81-3-5684-3298; e-mail junyosh{at}k.u-tokyo.ac.jp
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Abstract
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Background: Chemical speciation analysis is essential for the
biological monitoring of inorganic arsenic exposure using urine as
indicator medium. There is increasing demand for a certified reference
material (CRM) of urine matrix for arsenic speciation.
Methods: Urine (10 L) was collected from non-occupationally
exposed Japanese males. We prepared 954 bottles of urine, each
containing 
10 mL, after filtering and blending the urine stock. The
urine in each bottle was freeze-dried. Between-bottle homogeneity was
confirmed by measuring the concentrations of selected minor and trace
elements in the material and subsequent statistical analysis.
Certification was based on a collaborative analysis involving 15
laboratories.
Results: Certified values were determined for arsenobetaine
(0.069 ± 0.012 mg As/L), dimethylarsinic acid (0.036 ±
0.009 mg As/L), and total arsenic (0.134 ± 0.011 mg/L) as well as
for total selenium (0.059 ± 0.005 mg/L) and zinc (0.62 ±
0.05 mg/L), based on the analytical values from the collaborating
laboratories. Reference values are given for copper (0.010 mg/L) and
lead (0.0011 mg/L), based on definitive analysis at the National
Institute for Environmental Studies (NIES).
Conclusions: The present CRM, NIES CRM No. 18 Human Urine, is the
first human urine CRM for arsenic speciation and will be of value for
analytical quality assurance of the biological monitoring of arsenic
exposure.
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Introduction
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Adverse health effects arising from the consumption of drinking
water contaminated with inorganic arsenic from geological
sources is a serious problem in several regions of the world
(1). Assessment of arsenic exposure in these populations,
and for occupationally exposed persons, has been carried out by
analysis of urine because urinary excretion is the major pathway of the
elimination of arsenic from the mammalian body (2).
Inorganic arsenic (arsenite and arsenate) is methylated to less toxic
forms, i.e., methylarsonic acid
(MMA)1
and dimethylarsinic acid (DMA), in the body, and these four
species [As(III), As(V), MMA, and DMA] can be excreted in urine after
exposure to inorganic arsenic.
Humans are exposed to other forms of arsenic through the consumption of
seafoods. Arsenobetaine (AB) is the major form of the organic arsenic
ubiquitously found in edible fish and shellfish at concentrations up
to, and sometimes exceeding, 100 µg/g, and usually accounting for
virtually all of the arsenic present (3). Arsenosugars
(dimethylarsinoylribosides) are found in seaweeds at concentrations up
to 100 µg/g (3)(4). This group of organic
arsenic compounds is essentially nontoxic and is also excreted in
urine: AB is readily excreted in urine unchanged, whereas at least a
portion of the arsenosugars consumed appears to be excreted in the form
of DMA (5).
Speciation of arsenic compounds in urine is essential for the
biological monitoring of inorganic arsenic exposure. Several analytical
methods have been used for the accurate and precise speciation of
arsenic in urine. These usually are based on atomic spectrometry
preceded by the separation of arsenic species by the hydride generation
method or by liquid chromatography. Thus, techniques such as hydride
generation-atomic absorption spectrometry (HGAAS) and
HPLC-inductively coupled plasma mass spectrometry (HPLC-ICPMS)
have become routine because of high sensitivity and specificity.
The quality assurance of urine arsenic speciation analysis has recently
become a matter of concern. An intercomparison exercise has been
carried out using human urine with added inorganic arsenic,
MMA, and DMA as a test material (6). The preparation of a
candidate human urine reference material for arsenic speciation (AB and
DMA) has been reported (7).
We describe a newly available human urine Certified Reference
Material (CRM) for arsenic speciation, NIES CRM No. 18 Human Urine, and
its preparation and certification are also reported.
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Materials and Methods
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urine sample
Male clerical workers at the National Institute for Environmental
Studies (NIES), without known occupational exposure to arsenic
compounds, were asked to supply their first morning urine for 2
consecutive days in August 1996. Twenty-six workers supplied their
urine in 250-mL polypropylene bottles and delivered the urine to the
laboratory on the morning of the sampling. The urine was immediately
filtered through a 5 µm membrane filter to remove any debris and was
stored in a 20-L polycarbonate tank at -20 °C. A stock of 10 L
of urine was thus obtained.
preparation of the reference material
All bottles, tanks, and other apparatus used in the sampling and
preparation were cleaned by soaking overnight in 3 mol/L
HNO3 followed by vigorous rinsing with high
purity water (MQ).
The frozen urine was thawed and filtered through a 0.45 µm membrane
filter and stored in a second polycarbonate tank at 4 °C. The
filtration procedure took 2 working days, and on the next day 10-mL
portions of the stock were manually dispensed into 954 acid-washed,
numbered borosilicate glass bottles. The amount of the dispensed was
monitored by weight (10.00 ± 0.05 g). The stock urine was
continuously stirred with a Teflon propeller during the transfer of
urine to the bottles. The weight of bottle and dispensed urine was
recorded for every 50 bottle during this procedure to allow
calculation of weight loss by the subsequent freeze-drying. The bottles
were stored at -20 °C until being sent for freeze-drying by the
Wako Pure Chemical Co. Ltd. After freeze-drying, the headspace of each
bottle was filled with nitrogen, and the bottles were sealed with
Teflon-lined silicon caps secured with aluminum closures. The weight
loss from freeze-drying was 9.57 ± 0.02 g (n = 20).
Thus, the urine is reconstituted by the addition 9.57 g of pure
water to the bottle.
homogeneity assessment
Homogeneity of the prepared material was assessed by measuring the
concentrations of selected minor and trace elements in four subsamples
(0.5 g each) taken from each of six randomly selected bottles. Sodium,
magnesium, phosphorus, sulfur, potassium, calcium, and zinc
concentrations were measured by inductively coupled plasma atomic
emission spectrometry (ICP-AES) after the urine samples were digested
with nitric acid in a sealed Teflon bomb (8). Total arsenic
concentration was measured by ICPMS (HP-4500; Yokogawa Analytical
Systems Inc., Tokyo, Japan) with internal standardization (Y, for drift
correction) and matrix matching (for correction of spectroscopic and
nonspectroscopic interferences).
The concentrations obtained were statistically tested for
between-bottle variability by one way ANOVA using an SPSS
statistical program package (9).
collaborative analysis
After the homogeneity of the material was confirmed at NIES,
collaborative analysis for the certification of total arsenic and
arsenic species involving 10 laboratories was undertaken. Five other
laboratories provided analytical data other than total arsenic and
arsenic species. Two bottles were sent to each participant along with a
document describing how the material had been prepared and with
instructions for its handling. The participants were asked to provide
individual analytical results by their routinely-used methods for at
least five subsamples from one or both bottles. No extensive
collaborative analysis for other trace elements was organized.
certification
The mean and SD of the results from each participant were
calculated. If the SD was larger than expected for the analytical
method used and the concentration of the analyte, the value was
excluded from the certification procedure. A Grubbs test was then
applied to the remaining means to detect and reject outliers
(10). The mean of the remaining means was designated as the
Certified Value, and twice its SD was taken as the uncertainty range of
the Certified Value. This certification process has been used for all
NIES CRMs (11).
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Results and Discussion
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homogeneity
The results of the homogeneity assessment are shown in Table 1
. The mean element concentrations are given on a weight basis
(mg/kg), not on a volume basis (mg/L) because gravimetric sample
preparation was used at NIES to improve the ability to detect small
variability. The weight-based concentration can be converted to a
volume-based one using the specific density of the reconstituted
material (1.015 at 23 °C) measured at NIES.
Within-bottle relative standard deviation (RSD) was 0.4–3.4%
depending on the analytes. These values are in agreement with the
expected variability resulting from the preparation of the material and
the precision of ICP-AES and ICPMS measurements at the given
concentrations. The inherent variability derived from the preparation
of the material, i.e., the transfer of an aliquot of urine (10.00
± 0.05 g) into a bottle and the subsequent freeze-drying
(9.57 ± 0.02 g), was estimated to be 0.36%, taking the root
of the sum of the squares of the variabilities.
Between-bottle RSD was typically smaller than, or equal to,
within-bottle RSD; thus, no statistically significant variability was
revealed by ANOVA (Table 1
). A small, but not significant, variation
was observed for calcium, but the reason for this was not clear. From
these results, it can be concluded that the prepared material is
homogeneous at a practical level.
collaborative analysis for total arsenic
The total arsenic concentration in the material was determined in
five laboratories using six different analytical techniques (Table 2
). Because of the expected presence of AB in the material, which
is known to be resistant to acid digestion
(12)(13), those laboratories that used an
analytical method that depended on the chemical form of arsenic in the
sample were asked to use vigorous
HNO3/HClO4/H2SO4
digestion (12). Seven results, ranging from 0.130 to 0.146
mg/L, were reported from the five laboratories (laboratory 01 provided
three independent results), and none was rejected as an outlier. Thus,
the Certified Value for total arsenic was 0.137 ± 0.011 mg/L.
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Table 2. Analytical methods used for total arsenic analysis of
NIES CRM No. 18 Human Urine in collaborating
laboratories.
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collaborative analysis for arsenic speciation
Six laboratories provided arsenic speciation data (Table 3
). Some of the methods have been published elsewhere
(14)(15)(16)(17). It must be noted, however, that the detection
system used in the collaboration was almost exclusively ICPMS [one
laboratory used microwave-induced plasma mass spectrometry (MIP-MS)],
and the separation system used was exclusively liquid chromatography.
However, the separation principles differed from one laboratory
to another, i.e., cation-exchange, anion-exchange,
anion-exclusion, and reversed-phase chromatography were used.
Therefore, we considered it appropriate to determine the Certified
Value for AB and DMA based on the present collaborative analysis,
although the procedure was not consistent, in a strict sense, with NIES
CRM certification policy (11).
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Table 3. Chromatographic conditions used for the analysis of
arsenic species in NIES CRM No. 18 Human Urine in collaborating
laboratories.
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Eight results were provided by the six laboratories for AB and DMA. The
reported mean values were 0.0616–0.0776 mg As/L for AB (Fig. 1
) and 0.0295–0.0439 mg As/L for DMA (Fig. 2
), both ranges being rather more variable than the reported
values for total arsenic concentration. The CV of the means for AB was
<4% for all results, and the CVs for DMA were 0.9–6.8%. These CVs
demonstrated that the determination of AB and DMA in all of the
collaborating laboratories was highly repeatable. None of the means was
rejected by a Grubbs test. On the basis of these results, the Certified
Values for AB and DMA were 0.069 ± 0.012 and 0.036 ± 0.009
mg As/L, respectively.
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Figure 1. Individual analytical results from collaborating
laboratories for AB (mg As/L).
•, mean; bars, SD.
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Figure 2. Individual analytical results from collaborating
laboratories for DMA (mg As/L).
•, mean; bars, SD.
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Although MMA, As(III), and As(V) were detected in the material in most
of the laboratories, the results of their estimation were too variable
to determine Certified or Reference Values: reported values were
0.0022–0.0054 mg As/L for MMA, 0.0023–0.0071 mg As/L for As(III), and
0.0006–0.0114 mg As/L for As(V).
In four of the collaborating laboratories (laboratories 04, 06, 07, and
08), full separation and determination of arsenic species, including an
unidentified peak(s) on the chromatogram, was reported. Laboratories
04, 06, and 08 detected one unidentified peak, and laboratory 07
reported three. According to standard compounds used in the four
laboratories (Table 3
), the unidentified arsenic compound(s) was not
arsenocholine, tetramethylarsonium ion, or trimethylarsine oxide. The
sum of the arsenic species in the material was 0.112–0.129 mg/L in the
four laboratories, showing good agreement with the total arsenic
Certified Value (0.137 ± 0.011 mg/L).
stability of arsenic species in freeze-dried urine
Stability of the constituents in the freeze-dried urine was
assessed at NIES by measuring the concentrations of AB and DMA in
freshly reconstituted samples over 2 years for AB and 1 year for DMA.
The results are shown in Fig. 3
. No systematic trend was evident in the concentrations of AB
and DMA, and they were thus assumed to be stable under the storage
conditions used at NIES for the freeze-dried CRM (4 °C). NIES will
continue to monitor the stability of AB and DMA in this CRM.
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Figure 3. Stability of AB and DMA concentrations (mg As/L) in
freshly reconstituted NIES CRM No. 18 Human Urine.
Data points are means; bars, SD (n =
3 each). •, AB;  , DMA.
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Feldmann et al. (18) recently reported that As(III), As(V),
MMA, DMA, and AB concentrations in urine samples were stable for up to
2 months when stored at 4 °C and -20 °C. Changes the speciation
and total arsenic concentrations in some of the samples were reported
after longer storage periods. It is important to note that the
Certified Values of NIES CRM No. 18 are valid only for freshly
reconstituted material. However, reconstituted NIES CRM No. 18 is
assumed to be stable for up to 1 month when it is stored at <4 °C,
according to the results of Feldmann et al. as well as the results from
NIES and collaborative laboratories involved in the certification.
certified and reference values for other trace elements
Because the material was intended to be certified for total
arsenic and arsenic species, extensive collaborative analyses were not
organized for other constituents. Several collaborating laboratories
provided analytical results for other elements, and the results for
total selenium and zinc were satisfactory for determining Certified
Values in accordance with the NIES CRM certification policy
(11). Thus, taking into consideration the importance of the
two elements in nutritional research, NIES determined Certified Value
for selenium and zinc.
Nine results were reported for selenium from eight laboratories using
four different analytical methods: electrothermal vaporization atomic
absorption spectrometry (ETAAS), fluorimetry, instrumental neutron
activation analysis (INAA), and HGAAS. One result was rejected by a
Grubbs test, and those remaining were used for certification. The
Certified Value for selenium was 0.059 ± 0.005 mg/L.
Four results were available for zinc from three laboratories using
three different analytical methods: ICP-AES, isotope dilution ICPMS
(ID-ICPMS), and INAA. Although the number of results was rather small,
the four results were consistent and included definitive ID-ICPMS data.
Thus, the Certified Value for zinc was 0.62 ± 0.05 mg/L.
The concentrations of copper and lead were determined at NIES by
ID-ICPMS and ICPMS with solvent extraction and external calibration
(19). Consistent results were obtained, and therefore, the
results were considered reliable enough to be used as Reference Values.
The Reference Values for copper and lead were 0.010 and 0.0011 mg/L,
respectively.
comparison of arsenic species concentrations in crm with previously
reported data
The total arsenic concentration in this CRM, 0.137 mg/L, is in
good agreement with the value (0.131 ± 0.101 mg/L, uncorrected
for specific gravity) reported by Yamato (20) for 102
Japanese students (94 males and 8 females). The mean concentrations of
DMA and trimethylarsenic compound reported in that work were
0.0385 ± 0.0251 and 0.0754 ± 0.0976 mg/L, respectively, and
these values are in good agreement with the values for the present CRM
(Table 4
). Yamauchi et al. (21) reported that the urinary
total arsenic, DMA, and AB concentrations of 56 healthy Japanese (30
males and 26 females) were 0.129 ± 0.092, 0.0344 ± 0.0283,
and 0.0833 ± 0.0745 mg/L, respectively. It can thus be assumed
that the prepared CRM represents the arsenic species composition
typically found in the urine of the Japanese population.
In conclusion, a CRM for arsenic speciation has been prepared from
a filtered, blended stock of human urine. Its composition was
homogeneous enough to be used as a CRM. Certified Values were
determined for total arsenic, AB, and DMA concentrations. Certified
Values and Reference Values were given for other trace elements of
nutritional and toxicological importance. The concentrations of total
arsenic and arsenic species in this CRM are those typically found in
fish-eating populations, such as the Japanese, and this CRM is
particularly suitable for use in quality assurance of arsenic analysis
of urine from such populations.
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Acknowledgments
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The following participated in the collaborative analysis for
certification, and we deeply appreciate their efforts: K. Hanaoka,
National Fishery University; S. Himeno and K.Yuasa, Kitasato
University; S. Hirai, S. Suzuki, and Y. Okada, Musashi Institute of
Technology; Y. Inoue, Osaka City University; K. Jin, Hokkaido
Prefectural Institute of Public Health; T. Kaise, Tokyo University of
Pharmacy and Life Sciences; Y. Nakaguchi, Kansai University; T.
Narukawa, Chiba Institute of Technology; T. Sakai, Yokogawa Analytical
Systems Inc.; T. Shirasaki, Hitachi Science Systems Inc.; Y. Tamari,
Konan University; H. Tao and T. Nakazato, National Institute of
Resource and Environment; D.L. Tsalev, University of Sofia; C. Watanabe
and K. Miyazaki, University of Tokyo; and Y. Yoshioka, Shizuoka
University. We also thank C. Komatsu, C. Suzuki, K. Takata, and M.
Ukachi, NIES, for their contributions to the preparation of this CRM.
NIES CRM No. 18 Human Urine (2 bottles/unit) is available from
NIES. Address correspondence via e-mail (nies.crm@nies.go.jp).
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Footnotes
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1 Nonstandard abbreviations: MMA, methylarsonic acid; DMA, dimethylarsinic acid; AB, arsenobetaine; HGAAS, hydride generation-atomic absorption spectrometry; ICPMS, inductively coupled plasma mass spectrometry; CRM, Certified Reference Material; NIES, National Institute for Environmental Studies; ICP-AES, inductively coupled plasma atomic emission spectrometry; RSD, relative standard deviation; MIP-MS, microwave-induced plasma mass spectrometry; ETAAS, electrothermal vaporization atomic absorption spectrometry; INAA, instrumental neutron activation analysis; and ID, isotope dilution. 
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Abstract
Introduction
