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Human urine certified reference material for arsenic speciation.

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) [3] 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 [micro]g/g, and usually accounting for virtually all of the arsenic present (3). Arsenosugars (dimethylarsinoylribosides) are found in seaweeds at concentrations up to 100 [micro]g/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.

Materials and Methods


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 [micro]m membrane filter to remove any debris and was stored in a 20-L polycarbonate tank at -20[degrees]C. A stock of ~10 L of urine was thus obtained.


All bottles, tanks, and other apparatus used in the sampling and preparation were cleaned by soaking overnight in 3 mol/L HN[O.sub.3] followed by vigorous rinsing with high purity water (MQ).

The frozen urine was thawed and filtered through a 0.45 [micro]m membrane filter and stored in a second polycarbonate tank at 4[degrees]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 [+ or -] 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[degrees]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 [+ or -] 0.02 g (n = 20). Thus, the urine is reconstituted by the addition 9.57 g of pure water to the bottle.


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).


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.


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).

Results and Discussion


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[degrees]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 [+ or -] 0.05 g) into a bottle and the subsequent freeze-drying (9.57 [+ or -] 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.


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 HN[O.sub.3]/ HCl[O.sub.4]/[H.sub.2]S[O.sub.4] 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 [+ or -] 0.011 mg/L.


Six laboratories provided arsenic speciation data (Table 3). Some of the methods have been published elsewhere (14-17). 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).

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 [+ or -] 0.012 and 0.036 [+ or -] 0.009 mg As/L, respectively.

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 [+ or -] 0.011 mg/L).


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[degrees]C). NIES will continue to monitor the stability of AB and DMA in this CRM.


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[degrees]C and -20[degrees]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[degrees]C, according to the results of Feldmann et al. as well as the results from NIES and collaborative laboratories involved in the certification.




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 [+ or -] 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 [+ or -] 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.


The total arsenic concentration in this CRM, 0.137 mg/L, is in good agreement with the value (0.131 [+ or -] 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 [+ or -] 0.0251 and 0.0754 [+ or -] 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 [+ or -] 0.092, 0.0344 [+ or -] 0.0283, and 0.0833 [+ or -] 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.

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 (


(1.) National Research Council. Arsenic in drinking water. Washington, DC: National Academy Press, 1999:310pp.

(2.) Vahter ME. Arsenic. In: Clarkson TW, Friberg L, Nordberg GF, Sager PR, eds. Biological monitoring of toxic metals. New York: Plenum Press, 1988:303-21.

(3.) Edmonds JS, Francesconi KA. Arsenic in seafoods: human health aspects and regulations. Mar Pollut Bull 1993;26:665-74.

(4.) Shibata Y, Jin K, Morita M. Arsenic compounds in the edible alga, Porpyra tenera, and in nori and yakinori, food items produced from red algae. Appl Organomet Chem 1990;4:255-60.

(5.) Ma M, Le XC. Effect of arsenosugar ingestion on urinary arsenic speciation. Clin Chem 1998;44:539-50.

(6.) Crecelius E, Yager J. Intercomparison of analytical methods for arsenic speciation in human urine. Environ Health Perspect 1997; 105:650-3.

(7.) Cornelis R, Zhang X, Mees L, Christensen JM, Byrialsen K, Dyrschel C. Speciation measurements by HPLC-HGAAS of dimethylarsinic acid and arsenobetaine in three candidate lyophilized urine reference materials. Analyst 1998;123:2883-6.

(8.) Okamoto K, Fuwa K. Low-contamination digestion bomb method using a Teflon double vessel for biological materials. Anal Chem 1984;56:1758-60.

(9.) SPSS Inc. SPSS base 9.0.1 user's guide. Chicago, IL: SPSS, 1999.

(10.) Grubbs FE, Beck G. Extension of sample sizes and percentage points for significance tests of outlying observations. Technometrics 1972;14:847-54.

(11.) Okamoto K. Preparation, analysis and certification of PEPPERBUSH standard reference material. National Institute of Environmental Studies Research Report No. 18. Tsukuba, Japan: NIES, 1980.

(12.) Jin K, Ogawa H, Taga M. Study on wet digestion method for determination of total arsenic in marine organisms by continuous flow arsine generation and atomic absorption spectrometry using some model compounds. Bunseki Kagaku 1983;32:E171-6.

(13.) Le X-C, Cullen WR, Reimer KJ. Decomposition of organoarsenic compounds by using a microwave oven and subsequent determination by flow injection-hydride generation-atomic absorption spectrometry. Appl Organomet Chem 1992;6:161-71.

(14.) Shibata Y, Morita M. Speciation of arsenic by reversed-phase high performance liquid chromatography-inductively coupled plasma mass spectrometry. Anal Sci 1989;5:107-9.

(15.) Chatterjee A, Shibata Y, Yoshinaga J, Morita M. Application of a nitrogen microwave-induced plasma mass spectrometer as an element-specific detector for arsenic speciation. J Anal At Spectrom 1999;14:1853-9.

(16.) Hanaoka K, Tanaka Y, Nagata Y, Yoshida K, Kaise T. Watersoluble arsenic residues from several arsenolipids occurring in the twelve tissues of the starspotted shark Musterus manazo. Appl Organomet Chem;in press.

(17.) Sakai T, Inoue Y, Date Y, Aoyama T, Yoshida K, Endo G. Simultaneous speciation of anionic and cationic arsenic species by HPLC-inductively coupled plasma mass spectrometry with dual column and dual mode system. Appl Organomet Chem;in press.

(18.) Feldmann J, Lai VW-M, Cullen WR, Ma M, Lu X, Le XC. Sample preparation and storage can change arsenic speciation in human urine. Clin Chem 1999;45:1988-97.

(19.) Yoshinaga J, Morita M, Edmonds JS. Determination of copper, zinc, cadmium and lead in a fish otolith certified reference material by isotope dilution inductively coupled plasma mass spectrometry using off-line solvent extraction. J Anal At Spectrom 1999;14:1589-92.

(20.) Yamato N. Concentrations and chemical species of arsenic in human urine and hair. Bull Environ Contam Toxicol 1988;40:633-40.

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[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.

[3] 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.

* 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

Received August 10, 2000; accepted August 23, 2000.
Table 1. Results of homogeneity assessment of NIES CRM No. 18 Human

 Sodium Magnesium Phosphorus Sulfur

Mean Concentration 3190 105 946 817
 (n = 24), mg/kg
Within-bottle RSD, % 0.43 0.78 0.86 0.8
Between-bottle RSD, % 0.26 0.30 0.87 1.1
F-value 0.399 0.150 1.024 1.644
P 0.843 0.977 0.433 0.199

 Potassium Calcium Zinc Arsenic

Mean Concentration 1320 190 0.62 0.134
 (n = 24), mg/kg
Within-bottle RSD, % 0.47 0.42 2.1 3.4
Between-bottle RSD, % 0.60 0.68 2.3 1.3
F-value 1.609 2.609 1.264 0.149
P 0.208 0.061 0.322 0.978

Table 2. Analytical methods used for total arsenic analysis of
NIES CRM No. 18 Human Urine in collaborating laboratories.

Lab Analytical
ID Sample pretreatment n method Remarks

01 Pressurized HN[O.sub.3] 24 ICPMS Matrix matching,
 digestion Y as internal
 HN[O.sub.3]/HCl[O.sub.4]/ 4 ICP-HRMS (a) As(V) standard
 [H.sub.2]S[O.sub.4] addition
 HN[O.sub.3]/HCl[O.sub.4]/ 4 MIP-MS As(V) standard
 [H.sub.2]S[O.sub.4] addition
02 HN[O.sub.3]/HCl[O.sub.4]/ 6 ETAAS
 digestion, solvent
 extraction, solid-phase
 back extraction
03 HN[O.sub.3]/HCl[O.sub.4]/ 6 HGAAS
05 Reconstitution and 12 INAA
 re-freeze-drying an
10 HN[O.sub.3]/HCl[O.sub.4]/ 5 HGAAS

(a) HRMS, high-resolution mass spectrometry.

Table 3. Chromatographic conditions used for the analysis of arsenic
species in NIES CRM No. 18 Human Urine in collaborating laboratories.

ID LC column (a) Mobile phase Standard (b) Detector

01 LC-SCX (CE) 20 mmol/L pyridine, AS(III), As(V), ICPMS
 pH 2.60; 1.5 mL/min MMA, DMA, AB,
 LC-SCX (CE) 20 mmol/L pyridine, AS(III), As(V), MIP-MS
 pH 2.60, 1.5 mL/min MMA, DMA, AB,
04 C-18 (RP) 10 mmol/L As(III), As(V), ICPMS
 butane-1-sulfonic MMA, DMA, AB,
 acid, 4 mmol/L AC, TMAO
 hydroxide, 4 mmol/L
 malonic acid, 0.5
 ml/L methanol, pH
 3.0; 0.75 mL/min
06 Nucreosil 0.1 mol/L As(V), MMA, DMA, ICPMS
 100SA (CE) pyridine-formic AB, AC, TMAO,
 acid buffer, [TMA.sup.+]
 pH 3.1; 1.0 mL/min
 Nucleosil 0.02 mol/L ammonium As(V), MMA, DMA, ICPMS
 10OSB (AE) phosphate buffer, AB, AC, TMAO,
 pH 3.0; 1.5 mL/min [TMA.sup.+]

07 Gelpack 4.0 mmol/L phosphate As(III), As(V), ICPMS
 GL-IC-A15 + buffer, pH 2.66; MMA, DMA, AB,
 C75 (AE + 1.2 mL/min AC, TMAO,
 CE) [TMA.sup.+]
08 Shodex Rspak 5.0 mmol/L oxalic As(III), As(V), ICPMS
 NN-614 (CE) acid, 6.0 mmol/L MMA, DMA, AB,
 ammonium nitrate; AC, TMAO,
 1.0 mL/min [TMA.sup.+]
09 TSKgel Oapak-A 2 mmol/L sodium As(III), As(V), ICPMS
 (CE & Aex) carbonate, pH 3.8; MMA, DMA, AB
 1.0 mL/min
 SCR-102H (Aex) HN[O.sub.3], pH 2.1; As(III), As(V), HG-ICPMS
 1.5 mL/min MMA

(a) Separation mode in parentheses: CE, cation exchange; AE, anion
exchange; RP, reversed phase: Aex, anion exclusion.

(b) Standards used for the identification of arsenic compounds in
urine: AC, arsenocholine; TMAO, trimethylarsine oxide; [TMA.sup.+],
tetramethylarsonium ion.

Table 4. Certified and reference values for NIES CRM No.
18 Human Urine. (a)

 Certified Analytical
 Unit value methods (b)

Total arsenic mg/L 0.137 [+ or -] 0.011 a, b, c, d, e, f
AB mg As/L 0.069 [+ or -] 0.012 g, h
DMA mg As/L 0.036 [+ or -] 0.009 g, i
Total selenium mg/L 0.059 [+ or -] 0.005 d, e, f, j
Total zinc mg/L 0.62--0.05 f, k, l


Copper mg/L 0.010
Lead mg/L 0.0011

(a) The freeze-dried urine powder to be reconstituted with 9.57 g
of purified water. Specific gravity of the reconstituted urine was
1.015 (23[degrees]C).

(b) Analytical methods: a, ICPMS; b, ICP high-resolution mass
spectrometry (ICP-HRMS); c, MIP-MS; d, ETAAS; e, HGAAS; f, INAA;
g, HPLC-ICPMS; h, HPLC-MIP-MS; i, HPLC-HG-ICPMS; j, fluorimetry;
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Title Annotation:Drug Monitoring and Toxicology
Author:Yoshinaga, Jun; Chatterjee, Amit; Shibata, Yasuyuki; Morita, Masatoshi; Edmonds, John S.
Publication:Clinical Chemistry
Date:Nov 1, 2000
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