filtrates was divided by the total As concen­ tration in the colon filtrates, as measured by ICP­OES. The chromatographic recoveries for all colon digests, except for that of soil 4, were satisfactorily high: 93 ± 7% (mean ± SD) on average [the recovery of the soil 4 digest excluded; see Supplemental Material, Table 2 (doi:10.1289/ehp.0901794)]. Hence, most As species present in these digest super­ natants could be detected with the HPLC/ ICP­ MS protocol. Bioaccessibility calcula­ tions for these digests displayed the highest value (75.5%) for the iAsV­ incubated colon digest, whereas colon incubation of the con­ taminated soils resulted in As bioaccessibility values of 24% (soil 1), 44% (soil 2), and 36% (soil 3) (Table 3). In sharp contrast, As bio­ accessibility in the colon digest of soil 4 was only 0.3%. Even when taking into account the low chromatographic recovery of 15%, we obtained a low As bioaccessibility of 2.4%, which is still an order of magnitude lower than the bioaccessibility values for the other soil digests. Overall, colon bioaccessibility val­ ues (Table 3) for the four soils were consis­ tently lower than the corresponding intestinal bioaccessibility values (Table 1) obtained with the IVG method. The most important part of this study consisted of the As speciation analysis of the colon digests after the gastrointestinal incu­ bation of iAsV and the four contaminated soils. The original analytical protocol was optimized to detect the presence of iAsIII, iAsV, MMAIII, and MMAV. We detected an additional As species, MMMTAV, in many of the colon digests. We initially identified MMMTAV using a combination of reten­ tion­time matching and by fortifying the sample with the suspected standard using separation 1 with ICP­MS detection, but we used a second chromatography (separation 2) for ICP­MS and ESI­MS detection. Figure 1 shows HPLC/ICP­MS mass chromatograms of m/z 75 (75As) and HPLC/ESI­MS mass chromatograms of m/z 155 ([M­H] –) for an MMMTAV standard and a SHIME extract using separation 2. The retention times of the MMMTAV in the standard and MMMTAV in the sample were slightly offset because the matrix of the soil extract caused the decreased retention of MMMTAV on the C18 column. Tandem MS (MS/MS) of m/z 155 yielded a product ion of m/z 137 (loss of H2O) and, to a lesser extent, a product ion of m/z 121 (due to CH2AsO2–) and m/z 140 (loss of CH3). The molecular mass of 155 and cor­ responding fragments were consistent with other reports for MMMTAV (Yathavakilla et al. 2008). We detected significant As methylation upon colon incubation of 225 µg iAsV/L (Figure 2). The sum of the concentrations of MMAV (31.0 µg/L), MMAIII (4.5 µg/L), and HPLC-ESI-MS m/z = 155 Flow diverted from ESI-MS HPLC-ICP-MS m/z = 75 4 × 104 0 3 × 104 ) s t n u o c ( e c n a d n u b A ) S P C ( e c n a d n u b A 0 0 70 iAsv 600 Soil 1 250 Soil 3 60 L /s 50 e iec40 p s 30 s gA20 µ10 0 L500 / ise400 c pe300 s sA200 µg100 0 L200 / s ic150 e e p ss100 A µg50 0 Environmental Health Perspectives • volume 118 | number 7 | July 2010 1007 Soil Standard Standard Soil 14 ) s t n u o c ( e c n a d n u b 103 A 0 100 1 × 104 ) s t n u o c ( e c n a d n u b A 0 2 × 104 137 112 121 –H2O 140 155 130 137 –H2O 121 140 155 137 m/z 155 Soil Standard 155 125 150 m/z 175 Arsenic metabolism by human gut microbiota MMMTAV (43.7 µg/L) exceeded that of iAsV (39.0 µg/L) and iAsIII (34.8 µg/L). In contrast, iAs species were predominantly present in colon digests of soils 1, 2, and 3, whereas they were the only As species in the colon digest of slag soil 4 (Figure 2). The colon digest of soil 1 displayed a methylation percentage of 4.7% with MMAV (17 µg/L) and MMMTAV 800 Soil 2 700 /L600 s ie500 c pe400 s300 s gA200 µ100 0 iAsv 10 Soil 4 (slag) /L 8 s ice 6 e p ss 4 A µg 2 0 7 Time (min) iAsv iAsIII MMAv MMAIII MMMTAv 100 125 150 175 Figure 1. Identification of MMMTAV by HPLC/ESI-MS/MS and HPLC/ICP-MS (left) [using separation 2; see Supplemental Material (doi:10.1289/ehp.0901794)]. The gray and black traces in chromatograms represent analysis of a soil extract and MMMTAV standard, respectively. Right: MS/MS spectra of m/z 155 within each MS spectra. iAsv iAsIII MMAv MMAIII MMMTAv iAsIII MMAv MMAIII MMMTAv iAsv iAsIII MMAv MMAIII MMMTAv iAsv iAsIII MMAv MMAIII MMMTAv Figure 2. Concentration (mean ± SD) of chromatographically detected As species in colon digests of iAsV (225 μg/L) and of As-contaminated soils 1–4 (n = 3). Note the different scales for the As concentrations (y‑ axis) for iAsV and contaminated soils.