Improvements, Challenges, and the Investigation of Two Unknowns in Arsenic Urine Speciation Analysis by HPLC-ICP-MS: From Development to Post-implementation

Background: High-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICP-MS) is a critical analytical technique for determining the different forms of arsenic present in human urine. A new method was developed to overcome the shortcomings, such as poor resolution between critical arsenic species, of previously validated methods. The goal was to achieve resolution between 6 arsenic species that can be present in urine, including 2 common nontoxic organic species (arsenobetaine [AsB] and arsenocholine [AsC]), 2 methylated species with intermediate toxicity (monomethylarsonic acid [MMA] and dimethylarsinic acid [DMA]), and toxic inorganic forms (arsenous acid [AsIII] and arsenic acid [AsV]). In addition, efforts have been focused on identifying 2 unknown arsenic peaks present in approximately 5% of the patient population. Methods: HPLC-ICP-MS was developed using an anion-exchange analytical column with upfront dilution of urine samples. Some samples were reinjected into a high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer (QTOF-MS) to identify the unknown compounds. Results: The newly developed method achieved adequate resolution for 6 known arsenic species. The method was successfully validated after additional challenges, such as stabilizing the transformation of certain arsenic species (AsB and AsIII), were resolved. A retrospective analysis of 3050 urine samples revealed positivity rates of 79.1% and 54.7% for AsB and DMA, respectively. All the other species were detected ≤2% of the time. Two unknown arsenic species analyzed by HPLC-QTOF-MS were identified as dimethylarsinoyl acetic acid (DMAA), a possible arsenosugar metabolite, and p-arsanilic acid (ASA), a likely contaminant from preanalytical exposure to urinalysis test strips. Conclusions: This new method improves the reliability of arsenic speciation analysis in urine, benefiting both clinical laboratories and patients.