Spot urine samples were collected in polypropylene tubes, frozen within 1–2 hr of collection, shipped in dry ice, and stored from 8 to 18 years at − 70°C in the Penn Medical Laboratory, MedStar Research Institute (Washington, DC, USA). The freezers have been operating under a strict quality control system to guarantee secure sample storage. For arsenic analyses, urine samples were thawed in August 2007, and up to 1.0 mL was transferred to a small vial, transported on dry ice to the Trace Element Laboratory, Graz University, Austria, and stored at − 80°C until analysis. Urine samples were frozen for an average of 13 years (range, 8–18 years) before analysis.
We measured total urine arsenic concentrations (expressed on an elemental basis) using inductively coupled plasma/mass spectrometry (ICPMS). The limit of quantification for total urine arsenic was 0.1 μg/L. We checked measurement accuracy with 1+19 diluted human urine no. 18 from Japan’s National Institute of Environmental Studies (Tsukuba, Japan) (e.g., 0.5 mL urine + 9.5 mL water). The measured mean (± SD) total arsenic concentration of 144 ± 4 μg/L (n = 19) was in agreement with the certified concentration of 137 ± 11 μg/L. Total arsenic concentrations exceeded the limit of quantification in all samples.
Urine concentrations of arsenite, arsenate, MA, and DMA (expressed on an elemental basis) were measured using high-performance liquid chromatography/vapor generation ICPMS (Lindberg et al. 2006 (link)). The limits of quantification were 0.1 μg/L for arsenite and 0.5 μg/L for arsenate, MA, and DMA. Arsenite and arsenate were below the limit of quantification in 2 (1%) and 126 (70%) samples, respectively. MA and DMA exceeded the limit of quantification in all samples. The interassay coefficients of variation for an in-house reference urine sample for arsenite, arsenate, MA, and DMA were 3.8%, 4.5%, 4.3%, and 1.9%, respectively (n = 18). We did not detect thio-DMA, an arsenic species that has been related to arsenosugar exposure (Hansen et al. 2003 (link); Raml et al. 2005 (link)) and to high arsenic exposure in Bangladesh (Raml et al. 2007 (link)), in any of the study samples.
We measured urine arsenobetaine concentrations using cation-exchange chromatography on a Zorbax 300 SCX column (4.6 mm inner diameter × 250 mm; Agilent, Waldbronn, Germany) operated at 30°C. The mobile phase was 10 mM pyridine (pH 2.3, adjusted with formic acid) at a flow rate of 1.5 mL/min. We injected 20 μL of sample. The limit of quantification for urine arsenobetaine was 0.5 μg/L. We checked the accuracy of the measurements with 1+9 diluted human urine no. 18. The mean (± SD) measured value for arsenobetaine was 68 ± 2 μg/L (n = 18), in agreement with the certified concentration of 69 ± 12. Ninety-six (53%) samples had concentrations below the limit of quantification, reflecting infrequent seafood intake in the study population. The median (10th–90th percentiles) of urine arsenobetaine was 0.5 μg/L creatinine (< 0.5–137) [0.5 μg/g (< 0.5–6.1)]. Urine arsenobetaine concentrations were similarly low by region and other participant characteristics (data not shown).
We measured total urine arsenic concentrations (expressed on an elemental basis) using inductively coupled plasma/mass spectrometry (ICPMS). The limit of quantification for total urine arsenic was 0.1 μg/L. We checked measurement accuracy with 1+19 diluted human urine no. 18 from Japan’s National Institute of Environmental Studies (Tsukuba, Japan) (e.g., 0.5 mL urine + 9.5 mL water). The measured mean (± SD) total arsenic concentration of 144 ± 4 μg/L (n = 19) was in agreement with the certified concentration of 137 ± 11 μg/L. Total arsenic concentrations exceeded the limit of quantification in all samples.
Urine concentrations of arsenite, arsenate, MA, and DMA (expressed on an elemental basis) were measured using high-performance liquid chromatography/vapor generation ICPMS (Lindberg et al. 2006 (link)). The limits of quantification were 0.1 μg/L for arsenite and 0.5 μg/L for arsenate, MA, and DMA. Arsenite and arsenate were below the limit of quantification in 2 (1%) and 126 (70%) samples, respectively. MA and DMA exceeded the limit of quantification in all samples. The interassay coefficients of variation for an in-house reference urine sample for arsenite, arsenate, MA, and DMA were 3.8%, 4.5%, 4.3%, and 1.9%, respectively (n = 18). We did not detect thio-DMA, an arsenic species that has been related to arsenosugar exposure (Hansen et al. 2003 (link); Raml et al. 2005 (link)) and to high arsenic exposure in Bangladesh (Raml et al. 2007 (link)), in any of the study samples.
We measured urine arsenobetaine concentrations using cation-exchange chromatography on a Zorbax 300 SCX column (4.6 mm inner diameter × 250 mm; Agilent, Waldbronn, Germany) operated at 30°C. The mobile phase was 10 mM pyridine (pH 2.3, adjusted with formic acid) at a flow rate of 1.5 mL/min. We injected 20 μL of sample. The limit of quantification for urine arsenobetaine was 0.5 μg/L. We checked the accuracy of the measurements with 1+9 diluted human urine no. 18. The mean (± SD) measured value for arsenobetaine was 68 ± 2 μg/L (n = 18), in agreement with the certified concentration of 69 ± 12. Ninety-six (53%) samples had concentrations below the limit of quantification, reflecting infrequent seafood intake in the study population. The median (10th–90th percentiles) of urine arsenobetaine was 0.5 μg/L creatinine (< 0.5–137) [0.5 μg/g (< 0.5–6.1)]. Urine arsenobetaine concentrations were similarly low by region and other participant characteristics (data not shown).
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