Página principal

Yen-Ching Chen,1 Chitra J. Amarasiriwardena,2 Yu-Mei Hsueh,3 David C. Christiani1


Descargar 122.71 Kb.
Fecha de conversión18.07.2016
Tamaño122.71 Kb.

Manuscript number: EPI-040-2

2002/6/17



Stability of Arsenic Species & Insoluble Arsenic in Human Urine
Yen-Ching Chen,1 Chitra J. Amarasiriwardena,2 Yu-Mei Hsueh,3 David C. Christiani1

1Occupational Health Program, Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115, USA; 2Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115; 3School of Medicine, Department of Public Health, Taipei Medical College, Taipei, Taiwan
Running title: Stability of Arsenic Species & Insoluble Arsenic in Urine
Address correspondence to David C. Christiani, Occupational Health Program, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA. Telephone: (617) 432-3323. Fax: (617) 432-3441. E-mail: dchris@hohp.harvard.edu

Funding for the study was provided by the National Institute of Health grants ES 05947 and ES 00002.


Abbreviations used: Arsenite, As(III); arsenate, As(V); monomethylarsonic acid, MMA(V); monomethylarsonous acid, MMA(III); dimethylarsinic acid, DMA(V); arsenobetaine, AsB; high-performance liquid chromatogram inductively-coupled plasma mass spectrometry, HPLC-ICP-MS.
ABSTRACT

Urinary arsenic species are important short-term biomarkers that have been used in epidemiologic studies. However, the stability of soluble arsenic species and the amount of arsenic lost during sample pretreatment remain unclear. The objective of this study is to evaluate the stability of soluble arsenic species in urine and aqueous standards as well as to assess the amount of insoluble and soluble arsenic lost during pretreatment (centrifugation and filtration, respectively). HPLC-ICP-MS was used to speciate arsenic species (As(III), As(V), MMA, DMA, and AsB) in aqueous standards and in urine samples. The arsenic levels in both freshly collected urine samples (pH = 5.5 to 7.0) and NIST SRM 2670 toxic elements in frozen-dried urine (pH = 4.4) remained constant up to 6 months when stored at –20oC. In an aqueous solution mixed with 10 g/L of As(III), As(V), MMA and DMA standards and stored at 4 oC, As(III) and As(V) were stable only up to 4 weeks and MMA and DMA remained stable up to 4.5 months. The same phenomenon was observed for 100 g/L mixed aqueous standards. There was no significant loss of arsenic species in urine (<5%) when passed through a 0.45 m filter. The amounts of insoluble arsenic in urine lost during centrifuge ranged from 1/2 to 1/17 of soluble arsenic. These findings indicated that the urinary matrix plays an important role in stablizing arsenic species. Also, the loss of insoluble arsenic in urine during centrifuging results in underestimation of arsenic exposure, and may explain the lack of an association between arsenic exposure and the risk of health outcomes reported in some epidemiologic studies.


INTRODUCTION

Arsenic is ubiquitous in the earth’s crust and biosphere. Since the nineteenth century, arsenic has been widely used in the manufacture of glass, feed additives, pigment for analine dye, wallpaper, soap, medicine, wood preservatives, pesticides, metalloids, and semiconductor applications. Humans may be exposed to arsenic via ingestion, inhalation, or, to a less extent, skin absorption. Previous studies in humans have shown that exposure to arsenic may lead to cancer of liver, kidney, bladder, prostate, lymphoid, skin, lung, and colon, as well as to other adverse health effects (1-3). The reported order of arsenical toxicity reflected by carcinogenesis and vascular disorders is MMA(III)>As(III)>As(V)>MMA(V)=DMA(V) (4, 5). MMA(III) has a very short half-life and converts to MMA(V) in a short time (6); thus, it appears in trace amounts in urine and is difficult to measure. Hence, the MMA discussed throughout this paper refers to MMA(V).


Feldmann et al. (7) evaluated the stability of arsenic species in freshly collected urine samples. They found that the freshly collected urine samples were stable for at least 2 months when stored at temperatures under either 4℃ or -20℃. But scarce time points and lack of urinary pH information limited the reliability of their data. In addition,

several studies have assessed the stability of arsenic species in aqueous samples, but did not measure organic arsenic (MMA and DMA), and most studies lacked the urinary pH information (8-11). Some researchers (9, 10, 12) add HNO3 to stabilize the arsenic species. However, acidification of aqueous samples leads to changes of arsenic distribution immediately.


Urinary arsenic species are important short-term biomarkers and has been used in many epidemiologic studies. However, detailed stability information of organic arsenic in aqueous standards is lacking. In particular, no study has assessed the amount of insoluble arsenic in urine. The objective of this study is to assess the stability of soluble arsenic species in urine and aqueous standards, as well as the amount of insoluble and soluble arsenic lost during the sample pretreatment steps (centrifuge and 0.45 m filter, respectively) to improve a reliable exposure biomarker data for epidemiologic research.

EXPERIMENTAL METHODS

Speciation of Arsenic Species

The stock solution (1,000 μg/L) of As(III) was prepared by dissolving appropriate amounts of sodium arsenite (As(III), NaAsO2 , J. T. Baker, USA) in 4 g/L NaOH (Merck, USA). The 1,000 μg/L stock solutions of As(V), MMA, DMA, and AsB were prepared by dissolving appropriate amounts of disodium hydrogen arsenate heptahydrate (As(V), AsHNa2O4‧7H2O, Fluka, USA), monosodium acid methane arsonate (MMA, AsH4NaCO3, Chemical Service, USA), dimethylarsinic acid (DMA, C2H7AsO2, Fluka, USA), and arsenobetaine (CH3As+CH2COOH, Community Bureau of Reference, Geel, Belgium) in deionized water. All five stock solutions were stored at 4C in the dark and re-prepared every month. Stability of this storage method over several months has been confirmed (13). The working solutions were prepared daily by serial dilutions of the 1,000 g/L stock solutions with deionized water to give the following mixed calibration standards: 1, 10, 40, 100, and 150 g/L. A 20 g/L tellurium in 5% HNO3 prepared from 1,000 g/mL (Baker Instra-Analyzed Reagent, J.T. Baker, Phillipsburg, NJ, USA) was used as the internal standard.


An isocratic pump (Kontron HPLC pump 420) with an anion-exchange column (Hamilton PRP X-100 column, 4.1 mm I.D.  25 cm, 10 m particles, Hamilton, USA) and corresponding Hamilton guard column (2.3 mm I.D.  25 mm) was used for separation. The injection valve (Rheodyne Model 9125, Catati, CA, USA) with a 100-L injection loop was used for sample introduction. The HPLC mobile phase was a solution of 15 mmol/L tartaric acid (pH = 2.9, EM Science, USA) (14) at a flow rate of 1.5 mL/min. This solution was filtered through a 0.45 m filter (Waters, GHP Arcordisc Minispike, MA, USA) and degassed before use. A 10% HNO3 (nitric acid, Fisher, Optima, USA) solution was used to wash the nebulizer of ICP-MS between injections to prevent clogging. All glassware utilized for lab work was cleaned with 10% HNO3 (OmniTrace, Merck, USA). The ICP-MS used in this study was SCIEX ELAN 5000 (Perkin-Elmer, Norwalk, CT, USA) with a cross-flow nebulizer. Effluent from the column was mixed on-line with the internal standard via a zero-dead-volume mixing tee. The settings for the HPLC-ICP-MS system are listed in Table 1. The HPLC-ICP-MS was optimized by using a 10 g/L arsenic standard before sample analysis. Data from the HPLC-ICP-MS was imported into the ACD/Chrom Manager Package (Toronto, ON, Canada) and the deconvolution program was applied for peak area analysis.
Total Arsenic Analysis

Calibration standards for total arsenic determination were prepared from National Institute of Standards & Technology (NIST) Certified Standard (Gaithersburg, MD, USA) NIST 3103a arsenic standard solution. The NIST Standard Reference Material (SRM) 1643d (trace element in water) and NIST SRM 2670 (toxic metals in frozen-dried urine) were used as the calibration verification standard and quality-controlled standard for total arsenic determination, respectively. A solution of 250 g/L tellurium in 5% HNO3 was used as the internal standard for quantification of total arsenic. The total arsenic was determined by hydride-generation atomic-absorption-spectrometry (HGAAS) with detailed information described elsewhere (15).


Urine Sample Collection and Pretreatment

The urine samples were collected in polypropylene (PP) specimen containers and stored at –20C. Owing to the salt and organic content in urine, a 10-fold dilution was necessary to reduce the matrix effect. To measure the soluble arsenic in urine, samples were thawed at room temperature and then centrifuged at 3,000 g and 4℃ for 30 minutes. The precipitate was discarded, and the supernatant was passed through a 0.45 m filter (Waters, GHP Arcordisc Minispike, MA, USA) to remove particulate material.


Stability of Arsenic Species

The HPLC-ICP-MS was used to study the stability of 7 freshly collected urine samples from volunteers, the NIST SRM 2670 (normal level) standard solution, and 2 mixed arsenic aqueous standards (10 and 100 g/L of each arsenic species: As(III), As(V), MMA, and DMA). An AsB standard was not available when we did the stability test. The urine samples and arsenic standards were aliquoted into several microcentrifuge tubes and stored at –20 and 4°C, respectively. The pretreatment of urine samples is the same as described above. All urine samples were diluted for 10-fold and analyzed for arsenic species weekly throughout 6 months. In order to study the transformation between As(III) and As(V) and control for the interference from the other arsenic species in the solution, we repeated the above experiment using solutions with single species: 100 g/L of As(III) and 100 g/L of As(V).


Loss During Pretreatment

Loss during centrifugation. To measure insoluble arsenic, we collected 50 mL of urine and collected the precipitates after centrifuge. The precipitate was dissolved in 3 mL of 70% HNO3 (Mallinckrodt, NJ, USA) and then microwave digestion was applied. The amount of arsenic in precipitates was measured by HGAAS.
Loss during filtration. An 10 g/L arsenic aqueous standard (As(III), As(V), MMA, DMA, and AsB), a urine sample, and a spiked urine sample were used to evaluate the amount of arsenic lost during filtration. These solutions were separated into two aliquots, one remained untouched and the other was passed through a 0.45 m filter. The recovery of each arsenic species in the filtrate was then calculated. Because it is not plausible to inject an unfiltered urine sample directly into the HPLC column, we used filtered urine samples as the unfiltered ones and compared with re-filtered urine to measure the loss during filtration.

RESULTS

Speciation and Quantification

After reviewing the relevant literature, we found that most studies using a phosphate buffer had difficulties in separating AsB and As(III) and had problems with clogging in the nebulizer of ICP-MS (16). We modified Zheng et al.’s method (14) for speciation of arsenic species in water samples for urine sample analysis. We added an internal standard (20 g/L tellurium) and the NIST SRM 2670 arsenic urine standard to improve quantification. Factors taken into consideration for the selection of separation method include buffer solution, pH of the buffer, limit of detection of the method, and the linear range. The limits of detection in this study (Table 2) are either better than or similar to other reported methods (14, 16-25). The linear range for five arsenic species is 0 to 150 g/L, and we expect that most of the urine samples (diluted 10-fold) with arsenic would fall within this range (usually below 100 g/L).


To study the effect of pH on separation, we changed the buffer pH from 2.0 to 6.0. When pH<2.9, serious tailing of As(V) was observed. At the higher pH, all arsenic species started to overlap. The optimal pH for this analysis is 2.9. Although the buffer pH (2.9) is not close to that of human urine, the distribution of arsenic species stayed the same except at very high pH as shown by Zheng et al. (14). The effect of buffer concentration on the resolution of the arsenic species was also tested. We changed the buffer concentration from 5 to 30 mmol/L. The higher the buffer concentration, the faster the arsenic species eluted and started to overlap with each other (data not shown). Therefore, 15 mmol/L of buffer was applied throughout the experiment.
Limits of detection for As(III), As(V), MMA, DMA, and AsB by this method were 0.32, 0.25, 0.10, 0.29, and 0.10 g/L, respectively. The recovery of each species in the spiked (5 and 10 g/L of each five arsenic species) urine and aqueous sample were between 95 to 105%. Method parameters for arsenic speciation in urine by HPLC-ICP-MS are described in Table 2.
In addition, we analyzed five arsenic species in a NIST SRM 2670 elevated level and a normal level. Because no certified values for each arsenic species were available, the levels of arsenic in these two urine standards were compared with the results from other labs (Table 3). Our results are in agreement with data from other groups.
Stability of Arsenic Species

To control for the effects of MMA and DMA on distribution, we compared the stability of As(III) and As(V) in mixed solutions (As(III), As(V), MMA, and DMA) (Figure 1, Panel A & B) to the individual As(III) and As(V) aqueous standards (Figure 1, Panel C & D). We found that the presence of MMA and DMA did not affect the distribution of As(III) and As(V) in aqueous solution. But if the arsenic methylation enzymes (or specific microorganisms) are present, inorganic arsenic may be methylated to organic arsenic (e.g., MMA and DMA). The concentration of As(III) and As(V) species in the 10 g/L mixed arsenic aqueous standards were stable only up to the 29th day. The reduction of As(V) to As(III) began thereafter and was complete by day 36 (Figure 1, Panel A). We observed a similar effect, but at a slower rate with a 100 g/L standard: a significant change from As(V) to As(III) occurred after day 36, and was complete by day 94 (Figure 1, Panel B). For both aqueous standards, MMA and DMA concentrations remained stable up to 4.5 months since preparation. The 100 μg/L individual As(III) aqueous standard (pH = 6.0, 4℃) was stable for at least 3 months (Figure 1, Panel C). The rate of reduction of As(V) to As(III) in 100 μg/L As(V) individual aqueous standard (Figure 1, Panel D) is the same as in the mixed arsenic aqueous standard.


The average concentrations of 7 urinary arsenic species in freshly collected samples (pH = 5.5 to 7, -20℃) were stable over 6 months (Table 4, coefficient of variation (CV) for As(III): 2.4 to 19.4%, As(V): 2.8 to 37.5%, MMA: 5.1 to 13.1%, and DMA: 3.2 to 14.0%). The NIST SRM 2670 (normal level, pH = 4.4, -20℃) was stable at 6 months and the average concentration were: As(III): 2.5 ± 0.2 g/L, As(V): 1.5 ± 0.2 g/L , MMA: 8.3 ± 0.7 g/L, DMA: 47.2 ± 1.3 g/L, and AsB: 13.5 ± 0.9g/L (Figure 2).
Loss During Pretreatment

Loss during centrifuge. The amount of insoluble arsenic was analyzed from 4 urine samples. The ratio of soluble to insoluble total arsenic in urine ranges from 2 to 17 (Table 5).
Loss during filtration. For the 10 g/L arsenic mixed aqueous standard, the percentage loss of insoluble arsenic species after passing through the 0.45 m filter was within ± 5% for As(III), As(V), MMA, DMA, and AsB (Table 2).

DISCUSSION

We found that arsenic species in freshly collected urine (pH = 5.5 to 7) and SRM 2670 (pH = 4.4) remained stable up to 6 months when stored at temperatures -20℃ or less. In aqueous arsenic standards (stored at 4℃), MMA and DMA remain stable for at least 4.5 months, while As(V) reduces to As(III) within 4 weeks after preparation. The finding that stability of arsenic species in urine was longer than in the aqueous standards may be due to the complex matrix and pH of urine as well as the sample storage temperature. The amount of insoluble arsenic in urine lost during centrifuge is considerable for some urine samples and should be noted. This study provides new information on: 1) the loss of insoluble arsenic in human urine, 2) the stability of MMA and DMA in aqueous standards, and that DMA and MMA does not affect the distribution of inorganic arsenic, and 3) the importance of detailed monitoring of urinary arsenic species and pH information. If the amount of insoluble arsenic is also associated with health effects, then the discovery of insoluble arsenic in urinary precipitates provides possible clues to the heterogeneity seen in epidemiologic studies.


Stability of Arsenic Species in Urine

Feldmann et al. (7) evaluated the stability of arsenic species in freshly collected urine samples. They found that the freshly collected urine samples were stable for at least 2 months (under both 4℃ and -20℃), shorter than our observation of 6 months. The change of arsenic species among some of their subjects may be due to the few time points for sample measurement (1st, 2nd, 4th, 8th months), system change of the analytical machine, and failure to use internal standard to help quantification. Our study measured arsenic levels weekly, providing a better picture of the stability of arsenic species in both aqueous and urine samples. Moreover, Feldmann et al. did not record urinary pH values, an important factor for stabilizing arsenic species in both human urine and aqueous standards. The normal pH range of human urine is 4.5 to 8. We found a range in pH from 5.5 to 7 among the 7 urine samples collected and the sample pH did not change over 6 months.

It has long been known that As(V) and As(III) undergo interconversion in “aqueous” solution depending on the pH, temperature, oxygen content, light, and the presence of other substances. However, we did not find transformation between As(III) and As(V) in human urine (both freshly collected urine samples and SRM 2670), and only low concentrations of As(III) and As(V) were present in most freshly collected urine samples. The possible explanations for these phenomena include: 1) the complex matrix of urine stabilizes the distribution of arsenic species, and 2) most As(V) is reduced to As(III) in blood, then some of As(III) is methylated into less toxic forms (MMA and DMA), and finally, the remaining As(III) is retained in the keratin of skin, hair, or GI tract.
Stability of Arsenic Species in Aqueous Standards

Feldman (8) reported that As(III) completely disappeared from aqueous solution in about 4 days at 1 and 10 g/L levels, in about 7 days at 100 g/L level, and in about 18 days at the 1,000 g/L level under room temperature. Agemian (9), Aggett and Kriegman (10), and Hall et al. (12) tried to use HNO3 to stabilize the aqueous arsenic species. However, since the distribution of arsenic species changed right after acidification, this is not a preferable way of sample storage and thus was not tested in our study. Hall et al. (12) also reported that 0.5 and 5.0 g/L As(III) and As(V) aqueous standard could remain stable for at least 11 days when stored at 5°C. Because of the lower concentrations of arsenic in that study, a shorter stability of arsenic is expected. Their results are consistent with ours. They also found that 0.5 to 20.0 g/L As(V) was reduced to As(III) within 2 days if stored at room temperature. Usually, we preserve samples in portable freezers immediately after collection. Therefore, the stability of arsenic species under conditions of room temperature was not evaluated in our study. The studies discussed above (7-9) were limited in that they did not monitor organic arsenic (MMA and DMA) and lacked information on the urinary pH.


Loss During Centrifuge

One important finding is that the amount of insoluble arsenic in urine lost during centrifuge is 1/2 to 1/17 of soluble arsenic. Freshly collected urine samples usually formed precipitates after a couple of weeks of storage and the time varied between samples. The components of precipitation in urine may be changed by food intake, metabolism rate, and disease type, etc. Because of the complexity of precipitation, it is difficult to determine contents either by using the techniques of NMR (Nuclear Magnetic Resonance), X-ray diffraction or MS. A medical reference (26) reported that, for alkaline urine, amorphous phosphate salts and phosphates are possible constituents in the cloudy and milky precipitates, respectively, while in acidic urine, urates form the precipitates.


Loss During Filtration

The percentages of arsenic species in aqueous standards and urine samples lost during filtration are within a reasonable range (less than 5%). Therefore, the levels of soluble arsenic species of interest in the samples did not change after passing through a 0.45 m filter.


With our modifications, this HPLC-ICP-MS method becomes a powerful tool for urinary arsenic speciation, because we use tellurium as an internal standard to help quantification, and we use a NIST SRM 2670 and 1643d to compensate for any ionization efficiency changes and changes in the nebulization efficiency. The HPLC-ICP-MS method has been adopted for a decade; the results between different instruments for the same urine samples (18, 19, 27) are consistent and show that the HPLC-ICP-MS is a reliable method and should not affect the distribution of arsenic species. Because large amounts of chloride in urine may result in interference of argon chloride (ArCl) in HPLC-ICP-MS analysis, this possibility was evaluated by injecting 0.15M NaCl (the normal level in human urine). The result showed that chloride does not have significant interference on any of the 5 arsenic species studied.
Total urinary arsenic concentration as measured by HGAAS excludes insoluble arsenic. For subject 1 & 2 the summation of the level of total arsenic (supernatant) and total arsenic (precipitate obtained after centrifuge) was slightly higher than the total arsenic (original samples, without any pretreatment), indicating that all soluble arsenic had been measured and the small difference could be ignored. For subject 3 & 4, the summation of the level of total arsenic (supernatant) and total arsenic (precipitate obtained after centrifuge) is lower than the total arsenic (original samples, without any pretreatment). This result indicates that some insoluble arsenic could not be measured if no microwave digestion was used before analysis by HGAAS. The portable equipment, Arsenator, used to measure the current high-level arsenic exposure of drinking water in Bangladesh measures only soluble arsenic. Also, the simple filter system using activated alumina does not remove insoluble arsenic. Although the amount of insoluble arsenic might be lower in drinking water than in urine due to fewer precipitates, it may be important to develop a convenient tool to assess the role of insoluble arsenic in both urine and water in human health. In addition, the amounts of total soluble arsenic were consistently greater than the summation of four arsenic species (sum), indicating that there were other organic arsenic species that could be measured by the HGAAS method. Thus, it is important to differentiate the meanings of “total soluble arsenic” and “the sum of all arsenic species” for studies that attempt to relate arsenic methylation ability to a health outcome.
In summary, arsenic species in human urine is stable for at least 6 months, longer than inorganic arsenic in aqueous standards. The loss of arsenic species in both urine samples and aqueous standards during filtration is negligible. However, the amount of insoluble arsenic lost during centrifuge is noteworthy. Therefore, this report provides valuable information on an important biomarker—urinary arsenic species—for epidemiologic studies. Further research should include: monitoring the long-term stability of arsenic in urine samples (e.g., >1 year), the pH range that could maintain the distribution of arsenic species in urine; determine the contents in precipitates, and possibly to develop a standard method to measure insoluble arsenic in human urine.

Table 1. Settings of HPLC-ICP-MS for arsenic analysis

Instrument

Parameter

Settings












HPLC


Mobile phase (16)

Column


Injection volume
Flow rate


15 mmol/L Tartaric acid (EM Science, USA), use 14% NH4OH to adjust pH to 2.9
Anion-exchange column: Hamilton PRP X-100

(4.1 mm I.D.  25 cm, 10 m particles, Hamilton, USA)


100 L (Rheodyne Model 9125, Catati, CA, USA)
1.5 mL/min


ICP-MS

ICP system-

RF power


Sample and skimmer

Torch


Nebulizer

Spray chamber



1050 W (forward)

Nickel

Quartz


Cross-flow

Double-pass













Total arsenic analysis

Speciation of arsenic species


Gas flow rate (L/min)-

Plasma


Ar-Ar plasma

N2

Auxiliary

Nebulizer

Ar-Ar plasma
Data acquisition-

Dwell time (ms)

Sweeps per reading

Readings per replicate

Number of replicates

Points per spectral peak


Mass analyzed-

Analyte


Internal standard

14.85 Ar


0.15

0.8
0.85-0.95

200

6

1



5

1

75As



128Te (250 g/L)

15.00 Ar


0

0.8
0.85-0.95

100

1

1



2000

1

75As



128Te (20 g/L)


Table 2. Method parameters for arsenic speciation in urine by HPLC-ICP-MS

Arsenic Species

As(III)

As(V)

MMA

DMA

AsB

Retention time (min)


11.6

19.7

7.0

8.3

6.6

LOD (g/L)a


0.32

0.25

0.10

0.29

0.10

Absolute detection limit (ng)b


0.032


0.095

0.010

0.029

0.010

RSD (%)c


5.8

6.0

7.9

6.0

5.3

Linear range (g/L)


1.0-150

1.0-150

1.0-150

1.0-150

1.0-150

Slope of the calibration curve (counts/(sec×g/L))


29099

48457

53791

59895

14464

Correlation coefficient (R2) of the calibration curve


0.9999

0.9998

0.9998

0.9997

0.9995

Loss during filtration (%)d

10 g/L

Urine + 10 g/L

–2.73


1.65

1.83


-1.49

1.04


3.55

-1.33


4.46

1.26


2.73

Spiked Recovery (%)

5 g/L


10 g/L

101.3


98.7

98.5


103.2

103.7


101.9

99.2


102.8

100.2


102.6

a The limit of detection was calculated by 3 times of standard deviation (SD) of 10 measurements of the 0.1 g/L mixed arsenic aqueous standard.

b Injection volume is 100 L.

c The relative standard deviation (RSD) equivalent to reproducibility (coefficient of variation = 100% × SD/mean) was evaluated by 10 measurements of 2 g/L mixed arsenic aqueous standard.

d The 10 g/L arsenic aqueous standard was passed through the 0.45 m filter to see how much arsenic is retained on the filter.
Table 3. Determination of NIST Standard Reference Material 2670 of human urine by HPLC-ICP-MS




Arsenic species

Sum of species

Certified value

pH

Literature




As(III)

As(V)

MMA

DMA

AsB

SRM 2670 (g/L)

(Normal level)



2.5 ± 0.2

15.0 ± 3.3



<0.4

<2.5

1.5 ± 0.2

2.9 ± 0.7

6.9 ± 0.8

NDa



8.3 ± 0.7

9.5 ± 3.0

9.4 ± 0.9

16.0 ± 0.6



47.2 ± 1.3

48.2 ± 2.4

47.5 ± 1.6

52.1 ± 2.2



13.5 ± 0.9

21.2 ± 3.7

16.0 ± 1.1

15.5 ± 1.0



72.9 ± 1.9

96.8 ± 6.3

79.8 ± 2.3

84.4 ± 3.3


60


4.5

This work

Goessler (28)

Ritsems (29)

Zheng (14)




SRM 2670 (g/L)

(Elevated level)



3.2 ± 0.4

13.1 ± 4.5



<0.4

<2.5

397 ± 15

390 ± 52


354 ± 17

428 ± 12


8.6 ± 0.7

10.9 ± 2.1

9.2 ± 0.9

15.9 ± 0.8



47.4 ± 1.5

51.6 ± 3.4

50.7 ± 1.8

50.4 ± 1.9



13.5 ± 0.6

24.7 ± 0.7

16.2 ± 1.1

15.6 ± 0.4



489 ± 20

490 ± 52


430 ± 17

514 ± 14

480 ± 100


4.4

This work

Goessler (28)

Ritsems (29)

Zheng (14)



a ND, below detection limit.

Table 4. Six-month average level of arsenic species in freshly collected urine samples

(Storage temp = -20℃)

Subject

pH value

Six-month average level of arsenic species (μg/L)

As(III)

As(V)

MMA

DMA

1


5.5

2.60 ± 0.30

0.40 ± 0.15

4.37 ± 0.34

15.79 ± 0.50

2


7.0

0.31 ± 0.06

1.41 ± 0.14

3.27 ± 0.43

10.32 ± 0.72

3


6.0

1.29 ± 0.12

0.49 ± 0.23

4.08 ± 0.52

19.12 ± 0.93

4


5.5

1.06 ± 0.07

0.64 ± 0.28

1.54 ± 0.18

16.57 ± 0.82

5


6.0

1.90 ± 0.22

2.47 ± 0.07

1.68 ± 0.11

16.84 ± 0.93

6


5.5

0.45 ± 0.07

1.20 ± 0.20

1.56 ± 0.08

13.67 ± 0.90

7

6.0

1.26 ± 0.03

1.55 ± 0.12

2.32 ± 0.20

9.56 ± 1.34

* Repeated measure once per week throughout 6 months.

** Coefficient of variation (CV) for As(III): 2.4 to 19.4%, As(V): 2.8 to 37.5%, MMA: 5.1 to 13.1%, and DMA: 3.2 to 14.0%.



Table 5. Loss during centrifuge

Subject

No.


Supernatant (Soluble As, μg/L)




Precipitateb

(Insoluble As, μg/L)






Original samplec

(μg/L)





Ratio of

Soluble As/Insoluble As



As(III)

As(V)

MMA

DMA

Sum

Total Asa




Total As




Total As




1

0.53

0.53

0

8.38

9.44

13.99




7.27




16.24




1.92

2

0.09

0.65

0

7.16

7.90

8.24




3.67




10.37




2.25

3

0.82

1.68

1.66

14.60

18.76

27.31




4.57




40.62




5.98

4

0.44

1.11

0.47

7.47

9.49

22.88




1.31




37.04




17.47

a Arsenic level includes inorganic arsenic and other organic arsenic in addition to MMA and DMA analyzed by HGASS.

b Precipitate obtained after centrifuge, after microwave digestion, HGAAS was applied for analysis.

c No filtration or centrifuge applied and then HGAAS was used for analysis.


A



B

C

D





Figure 1. Stability of 10 g/L (Panel A) and 100 g/L (Panel B) arsenic aqueous standards (As(III), As(V), MMA, and DMA, pH = 6.0, Temp = 4℃) & 10 g/L (Panel C) and 100 g/L (Panel D) arsenic aqueous standards (As(III) and As(V), pH = 6.0, Temp = 4℃)
In Panel A, the level MMA and DMA keep stable throughout 4.5 months. The level of As(III) increased after a month since preparation; whereas the level of As(V) decreased after that. After As(V) reduced into As(III), the level of both species remain stable. The lower level of arsenic, the shorter time is needed for the conversion. Thus, this reaction was completed earlier than 100 g/L arsenic aqueous standards.
In Panel B, the level MMA and DMA keep stable throughout 4.5 months. The level of As(III) increased after a month since preparation; whereas the level of As(V) decreased after that. After As(V) reduced into As(III), the level of both species reached stable after 3 months since preparation.
In Panel C, 100 g/L As(III) aqueous standard was used to test if As(III) will oxidize into As(V) under pH = 6.0, Temp = 4℃. As(III) did not oxidize into As(V) throughout 3 more months.
In Panel D, 100 g/L As(V) aqueous standard was used to test if As(V) will reduce to As(III) under pH = 6.0, Temp = 4℃. As(V) reduced to As(III) after a month since preparation and the reaction was completed after 3 months since preparation.



Figure 2. Stability of NIST SRM 2670 (normal level) (As(III): 2.5 ± 0.2 g/L, As(V): 1.5 ± 0.2 g/L , MMA: 8.3 ± 0.7 g/L, DMA: 47.2 ± 1.3 g/L, and AsB: 13.5 ± 0.9g/L) ( pH = 4.4, Temp = -20℃). The level of all 5 arsenic species remained stable during the experiment.
REFERENCES AND NOTES

1. Chen CJ, Wang CJ. Ecological correlation between level in well water and age-adjusted mortality from malignant neoplasms. Cancer Res 50:5470-5474(1990).

2. Chen CJ, Chuang YC, You SL, Lin TM, Wu HY. A retrospective study on malignant neoplasms of bladder, lung and liver in blackfoot disease endemic area in Taiwan. Br J Cancer 53:399-405(1986).

3. Wu MM, Kuo TL, Hwang YH, Chen CJ. Dose-response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol 130:1123-1132(1989).

4. Lin TH, Huang YL, Wang MY. Arsenic species in drinking water, hair, fingernails, and urine of patients with blackfoot disease. J Toxicol Environ Health 53:85-93(1998).

5. Yamauchi H, Fowler BA. Ch.3: Toxicity and metabolism of inorganic and methylated arsenicals. In: Arsenic in the environment. Part II : Human health and ecosystem effects (Nriagu JO, ed). New York:John Wiley & Sons, Inc., 1994;35-53.

6. Le XC, Ma M, Lu X, Cullen WR, Aposhian HV, Zheng B. Determination of monomethylarsonous acid, a key arsenic mehtylation intermediate, in human urine. Environmental Health Perspectives 108:1015-1018(2000).

7. Feldmann J, Lai VW-M, Cullen WR, Ma M, Lu X, Le XC. Sample preparation and storage can change arsenic speciation in human urine. Clinical Chemistry 45:1988-1997(1999).

8. Feldman C. Improvements in the arsine accumulation - helium glow detector procedure for determining traces of arsenic. Anal Chem 51:664-669(1979).

9. Cheam V, Agemian H. Preservation of inorganic arsenic species at microgram levels in water samples. Analyst 105:737-743(1980).

10. Aggett J, Kriegman MR. Preservation of arsenic(III) and arsenic (V) in samples of sediment interstitial water. Analyst 112:153-157(1987).

11. Turner R. Oxidation state of arsenic in coal ash leachate. Environ Sci Tech 15:1062-1066(1981).

12. Hall GEM, Pelchat JC, Gauthier G. Stability of inorganic arsenic(III) and arsenic(V) in water samples. J. Anal. At. Spectrom. 14:205-213(1999).

13. Guerin T, Astruc M, Batel A, Borsier M. Multielemental speciation of As, Se, Sb and Te by HPLC-ICP-MS. Talanta 44:2201-2208(1997).

14. Zheng J, Kosmus W, Pichler-Semmelorock F, Kock M. Arsenic speciation in human urine reference materials using high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection. J Trace Elem Med Biol 13:150-156(1999).

15. Hsueh YM, Huang YL, Huang CC, Wu WL, Chen HM, Yang MH, Leu LC, Chen CJ. Urinary levels of inorganic and organic arsenic metabolites among residents in an arseniasis-hyperendemic area in Taiwan. Journal of Toxicology and Environmental Health, Part A 54:431-444(1998).

16. Saverwyns S, Zhang X, Vanhaecke F, Cornelis R, Moens L, Dams R. Speciation of six arsenic compounds using high-performance liquid chromatography --inductively coupled plasma mass spectrometry with sample introduction by thermospray nebulization. J Anal At Spectrom 12:1047-1052(1997).

17. Guerin T, Astruc A, Astruc M. Chromatographic ion-exchange simultaneous seperation of arsenic and selenium species with inductively coupled plasma-mass spectrometry on-line detection. J Chromatogr Sci 35:213-220(1997).

18. Le XC, Ma M. Speciation of arsenic compounds by using ion-pair chromatography with atomic spectrometry and mass spectrometry detection. J Chromatogr A 764:55-64(1997).

19. Le XC, Cullen WR, Reimer KJ. Human urinary arsenic excretion after one-time ingestion of seaweed, crab, and shrimp. Clinical Chemistry 40:617-624(1994).

20. Le XC, Ma M. Short-column liquid chromatography with hydride generation atomic fluorescence detection for the speciation of arsenic. Anal Chem 70:1926-1933(1998).

21. Kavanagh P, Farago ME, Thornton I, Goessler W, Kuehnelt D, Schlagenhaufen C, Irgolic KJ. Urinary arsenic species in Devon and Cornwall residents, UK. A pilot study. Analyst 123:27-29(1998).

22. Larsen EH, Pritzl G, S.H. H. Speciation of eight arsenic compounds in human urine by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection using antimonate for internal chromatographic standardization. J Anal At Spectrom 8:557-563(1993).

23. Moldovan M, Gomez MM, Palacios MA, Camara C. Arsenic speciation in water and human urine by HPLC-ICP-MS and HPLC-MO-HG-AAS. Microchemical Journal 59:89-99(1998).

24. Le XC, Cullen WR, Reimer KJ. Speciation of arsenic compounds by HPLC wtih hydride generation atomic absorption spectrometry and inductively coupled plasma mass spectrometry detection. Talanta 41:495-502(1994).

25. Story WC, Caruso JA, Heitkemper DT, Perkins L. Elimination of the chloride interference on the determination of arsenic using hydride generation inductively coupled plasma mass spectrometry. J Chromatogr Sci 30:427-432(1992).

26. Beers MH, Berkow R. The Merck Manual of Diagnosis and Therapy. Whitehouse station, NJ:Merck research laboratories, 1999.

27. Crecelius E, Yanger J. Intercomparison of analytical methods for arsenic speciation in human urine. Environ Health Perspect 105:650-653(1997).

28. Goessler W, Kuehnelt D, Irgolic J. Determination of arsenic compounds in human urine by HPLC-ICP-MS. In: Arsenic: Exposure and Health Effects (Abernathy CO, ed). London:Chapman & Hall, 1997;33-44.

29. Ritsema R, Dukan L, i Navarro TR, van Leeuwen W, Oliveira N, Wolfs P, Lebret E. Speciation of arsenic compounds in urine by LC-ICP MS. Appl Organometallic Chem 12:591-599(1998).







La base de datos está protegida por derechos de autor ©espanito.com 2016
enviar mensaje