Optimization of method conditions for Arsenic speciation using isocratic HPLC coupled with ICP-MS Indranil Sen, Wei Zou, 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 Background: Arsenic is toxic and even carcinogenic in humans depending on the chemical nature and oxidation states of the compounds in various physiological concentrations. In the body, arsenic is metabolized to several different inorganic and organic species, including trivalent (As-III) and pentavalent (As-V), and the methylated species such as Monomethylarsonic acid, (MMA), and Dimethylarsinic acid (DMA), in addition to Arsenobetaine (AsB) and Arsenocholine (AsC) which stays unaffected in the body. The Arsenic species are excreted through urine. Urine is used as the matrix to measure the internal dose of Arsenic species. In order to determine the level of toxic exposures to human it is very important to ascertain the concentration of each of Arsenic species to identify the background contribution of inorganic and organic Arsenic species exposure. Analytical Method: A reliable and sensitive method to measure the concentration of six Arsenic compounds in humans is essential for exposure assessment and eventually to understand how human health might be affected by exposure to Arsenic. The test principle utilizes human urine, diluted 1:10 into the diluent buffer 10 mM Potassium Phosphate, monobasic, 2% Methanol, pH 5.8. Mobile phase used for the analysis was a mixture of 5mM Ammonium Phosphate, dibasic buffer and 5mM Ammonium Nitrate, pH 9.0, 2% Methanol, with 20 µg/L of Germanium used as internal standards1. The separation is performed with Hamilton PRP-X100® anion exchange column using Agilent 1200 isocratic HPLC coupled with Agilent 7700 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) as a detector2. The reporting limits are approximately 1 ppb for biomonitoring samples. 2. Optimization of method conditions for six Arsenic species: Health effects linked to Arsenic exposure: 1. Analysis of Arsenic species in human urine (NIST SRM 2669): Instrumentation: Objective: Develop and validate a method to measure toxic Arsenic compounds in urine samples by using high pressure liquid chromatography coupled with Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS). Method Challenges: The baseline resolved separation of Arsenobetaine (AsB) with As-III is critical since in real urine sample AsB could present in 100 ppb range with very low As-III (1-2 ppb) concentration. Besides, As-III tends to oxidize at room temperature to As-V during the course of long analysis. Solutions: The baseline resolved separation of Arsenobetaine (AsB) with As-III was achieved by trying various mobile phase combinations as well as the right pH conditions. 2. Stability of As-III species, was a major analytical challenges. In order to prevent the oxidation of As-III, we introduced several steps including the following: i) a pH 5.8 sample preparation buffer ii) removal of oxygen present in the mobile phase by degassing with Argon and iii) use of low temperature (-70ºC) for storage and iv) flip cap airtight tubes for long term storage. 2. Analysis of Arsenic species in human urine (GEQUAS Round 48 Proficiency Test).: Why we need to do speciation? Conclusion: We have validated a stable isocratic method to measure six Arsenic species in urine. We have achieved good baseline separation of Arsenobetaine (AsB) and As-III and we are able to analyze As-III at 1 ppb concentration consistently. 1. Optimization of method conditions for six Arsenic species: Toxicity differs in chemical species of Arsenic: 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. Acknowledgement: This work was partially funded by CDC Cooperative Agreement (5U38EH000481-03) supporting the California Environmental Contaminant Biomonitoring Program.