New Agilent HPLC column for routine determination of arsenic species in human urine by HPLC-ICP-MS

Background:

While arsenic is often considered to be the synonym for toxin, arsenic toxicity depends strongly on the species being present. Humans are exposed primarily through ingestion via diet (drinking water, seafood) or inhalation and workplace exposure. Arsenic is used in the manufacture of glass, pigments, medicinals, pesticides, wood preservation and semiconductor products. Once ingested, arsenic gets metabolised to a certain degree depending on speciation end level of exposure and is predominantly excreted in the urine. Therefore urinary measurement is an excellent biomarker for As speciation and differentiation of arsenic species is valuable information to trace the route of exposure. However various obstacles have prevented the speciation analysis of arsenic in urine from becoming routine:

• finding chromatographic conditions that allow the determination of the main As species in one run without changing columns (anionic, cationic, etc.)
• achieving good reproducible separation with high sensitivity and dynamic range without complicated gradient conditions
• resolving or eliminatimng the ArCl interference on As which is derived from the high NaCl concentration present in urine
• aciving long time stability despite the high total dissolved solids (TDS) resulting from the sample matrix and HPLC buffer systems.

The new column material:
Since none of the various commercially available columns proved 100% satisfactory at meeting the Agilent application goals, a new column was developed and manufactured (Agilent G3288-8000, 4.6 x 250  mm plus G3154-65002 Guard column). The new column provides excellent resolution of As(III) from both arsenobetaine and DMA, as well as good separation of MMA from chloride. Together with the guard column, this material allows for the analysis of indiluted 5-µL aliquots of human urine samples.

Mobile phase:
In order to buffer the changing matrix between different urine samples but also avoid excessive total salt concentrations that would compromise long term stability, a mixture of sodium acetate and sodium nitrate was used as the mobile phase. For enhancing the sensitivity, 1% of ethanol was added as a modifier.

Results:


All five main arsenic species present in human urine (As(III), As(V), AB, MMNA, DMA) were resolved from each other and from chloride within a separation time of 12 min. Detection limits were excellent and ranged from 0.05 to 0.1 µg/l depending on species. Reproducibility of peak area was better than 3% at 10 µg/L and reproducibility of the retention time was better than 0.6%. The arsenic species determined in the CRM NIES No. 18 agreeed well with the certified values vor DMA and AB.

The full report can be downloaded from Agilent's server as an application note:

Tetsushi Sakai, Steve Wilbur, Routine Analysis of Toxic Arsenic Species in Urine Using HPLC with ICP-MS, Agilent Application Note, August 25, 2006, 5989-5505EN


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January 10, 2023: Online addition of an internal standard for high performance liquid chromatography-inductively coupled plasma mass spectrometry

last time modified: March 8, 2024