The Separation of Dimethylarsinic Acid, Methylarsonous Acid, Methylarsonic Acid, Arsenate and Dimethylarsinous Acid on the Hamilton PRP-X100 Anion-Exchange Column Ju È rgen Gailer, 1 * Sean Madden, 2 William R. Cullen 3 and M. Bonner Denton 2 1 Department of Molecular and Cellular Biology, The University of Arizona, Life Sciences South Building, Tucson, AZ 85721, USA 2 Department of Chemistry, The University of Arizona, Tucson, AZ 85721, USA 3 Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1 In order to separate the potential arsenite metabolites methylarsonous acid and dimethyl- arsinous acid from arsenite, arsenate, methyl- arsonic acid and dimethylarsinic acid, the pH- dependent retention behaviour of all six arsenic compounds was studied on a Hamilton PRP- X100 anion-exchange column with 30 mM phos- phate buffers (pH 5, 6, 7, 8 and 9) containing 20% (v/v) methanol as mobile phase and employing an inductively coupled plasma atomic emission spectrometer (ICP–AES) as the ar- senic-specific detector. Baseline separation of dimethylarsinic acid, methylarsonous acid, methylarsonic acid, arsenate and dimethylar- sinous acid was achieved with a 30 mmol dm 3 phosphate buffer (pH 5)–methanol mixture (80:20, v/v) in 25 min. Arsenite is not baseline- separated from dimethylarsinic acid under these conditions. Copyright # 1999 John Wiley & Sons, Ltd. Keywords: arsenite; arsenate; methylarsonic acid; dimethylarsinic acid; methylarsonous acid, dimethylarsinous acid; speciation; HPLC–ICP– AES Received 25 February 1999; accepted 30 April 1999 INTRODUCTION After the ingestion of inorganic arsenic [As(III) and As(V)] by humans, methylarsonic acid and di- methylarsinic acid together with unchanged inor- ganic arsenic have been detected in human urine. 1–4 Recently, however, chronic exposure of rats to drinking water containing arsenite, methylarsonic acid or dimethylarsinic acid for seven months and subsequent analysis of urine by HPLC coupled on- line to an inductively coupled plasma mass spectro- meter (ICP–MS) revealed two unidentified, prob- ably novel, arsenic metabolites. 5 The molecular identification of these peaks brings about the need to develop HPLC methods that are capable of separating potential new metabolites from those that are already known. Although methylarsenicals are commonly found in the environment, these are usually reported to be As(V) derivatives, largely because the most commonly used analytical methodologies, such as hydride generation under acid conditions, are poorly capable of making the distinction between As(III) and As(V). The proposed pathway for the enzymatic methylation of arsenite in mammals involves its oxidative methylation to methylarso- nate, 6 with S-adenosylmethionine being the likely methyl donor. 4,7,8 After subsequent reduction of methylarsonate [As(V)] to methylarsonous acid [As(III)]—which may be accomplished chemically by the endogenous thiol glutathione (GSH) 9,10 — methylarsonous acid is then oxidatively methylated APPLIED ORGANOMETALLIC CHEMISTRY Appl. Organometal. Chem. 13, 837–843 (1999) Copyright # 1999 John Wiley & Sons, Ltd. CCC 0268–2605/99/110837–07 $17.50 * Correspondence to: Ju ¨rgen Gailer, Department of Molecular and Cellular Biology, The University of Arizona, Life Sciences South Building, Tucson, AZ 85721, USA. Contract/grant sponsor: Austrian Fonds zur Fo ¨rderung des wis- senschaftlichen Forschung; Contract/grant number: J01303-CHE. Contract/grant sponsor: Thermo Jarrel Ash Corporation. Contract/grant sponsor: Superfund Basic Research Program NIEHS (National Institute of Environmental Health Sciences; Contract/ grant number: ES-04940. Contract/grant sponsor: Southwest Environmental Health Sciences Center; Contract/grant number: P30-ES-06694.