Concentrations of arsenic in human urine: A correlation between total arsenic by ICP-MS and speciated arsenic by HPLC-ICP-MS Indranil Sen, Wei Zou, Josephine Alvaran, Linda Nguyen, Ryszard Gajek, Jianwen She* Biochemistry Section, Environmental Health Laboratory Branch California Department of Public Health, Richmond, CA 94804 *Corresponding author: jianwen.she@cdph.ca.gov Analytical Objective: We developed and validated an analytical method for the quantitative determination of total arsenic by using inductively coupled plasma mass spectrometry (ICP-MS). We have also developed and validated a method to measure individual arsenic compounds (AsC, AsB, As-III, DMA, MMA, As-V) in urine samples by using high pressure liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Arsenic results from both analytical methods were compared. Background: Arsenic is a widespread environmental contaminant. Adverse human health effects depend on the chemical form of arsenic, exposure levels, duration in the body and other factors. Arsenic can be ingested as one or more species, and species can be metabolized into several different inorganic and organic forms. Arsenic species are excreted through urine, which is routinely used as the biological matrix to measure the internal dose of arsenic species. Not all arsenic species are associated with adverse health effects. Therefore, in order to evaluate exposure to arsenic species of concern, it is very important to ascertain the urinary concentrations of each arsenic species. Figure 1 shows some environmental sources of arsenic exposure. Figure 2 illustrates bioaccumulation of arsenic along the food chain. Figure 3 shows relative arsenic species LD50 values based on a rat study. Figure 1: Environmental sources of arsenic exposure Table 1: Relative arsenic species LD50 values based on a rat study3,4,5 Figure 2: Bioaccumulation of arsenic along the food chain Figure 4: HPLC-ICP-MS Method Effectively Separates Six Arsenic Species Standards Materials and Methods: Analytical Method: ICP-MS for total arsenic concentration Sample Preparation: The samples were prepared by diluting human urine (1:20) with 4500 µL of diluent, 250 µL of 2% nitric acid, and 250 µL of urine sample in 15 mL conical tubes (brand and lots prescreened for contaminants). Diluent: The diluent consisted of 2% nitric acid, 0.2% sulfamic acid, 1000 µg/L gold standard solution, 1.5% ethanol, 0.0005% Triton X-100, 50 µg/L germanium (Ge), 50 µg/L rhodium (Rh) and 60 µg/L rhenium (Re). (Note: For this method multiple metals are analyzed concurrently with total arsenic.) ICP-MS Parameters: An Agilent 7500 CE ICP-MS equipped with a micromist nebulizer and helium collision cell was used. Typical values were as follows: RF power 1550 watts; RF matching 1.89 V; Sample depth 8.0 mm; torch H 0.4 mm, torch-V 0 mm, carrier gas flow 0.96 L/min; makeup gas: 0.15 L/min; nebulizer pump 0.1 rps; spray chamber temperature 2 ºC, extraction lens 1: 0 V; extraction lens 2: -125V; omegas bias ce-24V; omega lens 0.2 V; cell entrance -30 V; cell exit -30 V; deflect 13.6 V; plate bias -50 V; He flow (for collision cell) 4.6 mL/min; H2 gas 3 mL/min; octapole RF 180 V, octapole bias -4 V; Energy Discrimination 8.0 V. Figure 5: HPLC-ICP-MS Method Effectively Separates Arsenic Species in Human Urine 6. As-V Results: Urine samples from 26 anonymous adult donors were collected and analyzed. A correlation between results from the sum of arsenic species determined by arsenic speciation analyses and results from total arsenic analyses are shown in Figure 6. Figure 6: Correlation between the sum of arsenic species and total arsenic data of urine samples collected from anonymous adult donors B) Analytical Method: HPLC-ICP-MS for arsenic speciation Sample Preparation: The samples were prepared by diluting human urine (1:10) with 10.0 mM potassium phosphate monobasic buffer at pH 5.8. Separation: A Hamilton PRP-X100® anion exchange column using an Agilent 1200 HPLC pump coupled with an Agilent 7700 ICP-MS was used as the detector. Mobile phase: The mobile phase consisted of a mixture of 5.0 mM ammonium phosphate dibasic buffer, 5.0 mM ammonium nitrate, pH 9.0, with 2% methanol, and 20 µg/L of germanium (Ge) as the internal standard. ICP-MS Parameters: An Agilent 7700 CE ICP-MS equipped with a micromist nebulizer and a helium collision cell was used. Typical values were as follows: RF power 1550 watts; RF matching 1.80 V; sample depth 8.0 mm; carrier gas flow 1.07 L/min; nebulizer pump 0.3 rps; spray chamber temperature 2ºC, extraction lens 1: 6.4 V; extraction lens 2: -135V; omegas bias -65V; omega lens 9.4 V; cell entrance -40 V; cell exit -56 V; deflect 13.6 V; plate bias -50 V; He flow (for collision cell) 1.9-2.1 mL/min; octapole RF 160 V, octapole bias -8 V; Energy Discrimination 5.0 V. Slide Source: Lindemann et. al. Thermo-Fisher Scientific Conclusion: A simple, yet robust analytical speciation method was developed to measure six arsenic species in human urine using HPLC coupled with ICP-MS. Application of the method was demonstrated by analyzing a set of urine samples collected from anonymous donors. The sum of the 6 arsenic species values in each dornor’s urine is nearly equivalent to the total urinary arsenic measured by the ICP-MS method. A strong correlation between total urinary arsenic and the sum of arsenic species data was observed which exhibited the success of both analytical methods. Chemical Compound LD50 (mg/kg) Arsenous (III) Acid (As-III) 14 Arsenic (V) Acid (As-V) 20 Monomethylarsonic acid (MMA) 700-1800 Dimethylarsinic acid (DMA) 700-2600 Arsenocholine (AsC) >10,000 Arsenobetaine (AsB) C) Chromatograms Example chromatograms from arsenic speciation analyses are shown in Figures 4 & 5. The chromatogram in Figure 4 shows that each of the six arsenic species standards demonstrated good separation of all six arsenic species. Figure 5 shows that each of the six arsenic species was well separated for the anonymous donor urine data as well. (Note: Dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) are metabolites of inorganic arsenic species and may not always be detected in human urine). References: FDA Elemental Analysis Manual: Section 4.10: High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Determination of Four Arsenic Species in Fruit Juice, Version Draft 0.82 (August 2010), Author: Sean D. Conklin. (http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ElementalAnalysisManualEAM/ucm219640.htm) Carl P. Verdon et. al., Anal. Bioanal. Chem., 2009, 393, 939-947. Kaise, T., et al. Applied Organometallic Chemistry, 1989. 3(3): p. 273-277 Kaise, T., et al. Applied Organometallic Chemistry, 1992. 6(4): p. 369-373 Platanias, L.C. Journal of Biological Chemistry, 2009. 284(28): p. 18583-18587 Acknowledgement: This work was partially funded by CDC Cooperative Agreement (5U38EH000481-03) supporting the California Environmental Contaminant Biomonitoring Program.