Arsenic speciation by HPLC-ICP-MS

Urine samples (150 μL) were diluted × 10 with deionised water and As speciation was measured using high performance liquid chromatography coupled to ICP-MS (HPLC-ICP-MS) using the method described by Button et al.⁵⁷. In summary, a GP50 gradient pump and an AS auto-sampler (Dionex, USA) were coupled to the ICP-MS instrument with PEEK tubing. Chromatography was performed with a PRP-X100 anion exchange column and a PRP-X100 guard column (Hamilton, USA) using gradient elution with the mobile phase (pH 8.65, 1 mL/min) alternating between 4 and 60 mM NH₄NO₃. A 3-point calibration was used with 1, 10 and 50 As μg/L solutions of As^III and a mixed solution of 1, 10 and 50 As μg/L As^V, MA, DMA and AB. Figure 7 shows a standard chromatogram obtained for calibration solutions. The LODs for this method (3σ of blank values) are reported by Watts et al.⁵⁸: 0.8; 1.5; 0.7; 0.3; 1.3 As μg/L for As^III, As^V, MA, DMA and AB respectively. It is noted that this method cannot distinguish the trivalent and pentavalent forms of both MA and DMA which vary in genotoxicity⁵⁹.

Chromatograms obtained for standard calibration solutions at 1, 10 and 50 μg/ L. Calibration of arsenate (As^V), methylarsonate (MA), dimethylarsinate (DMA) and arsenobetaine (AB) was performed with mixed solutions of arsenic (V) oxide hydrate (As₂O₅·xH₂O), monomethylarsonic acid ((CH₃AsO(OH)₂), dimethylarsinic acid ((CH₃)₂AsO(OH)) and arsenobetaine ((CH₃)₃As + CH₂COO-) respectively. Calibration of arsenite (As^III) has been plotted simultaneously and was achieved with separate solutions of arsenic trioxide (As₂O₃).