Japanese Society of Grassland Science ISSN1744-6961 Grassland Science 53 (2007) 201–204 © 2007 Blackwell Publishing Ltd 201 Blackwell Publishing Asia ORIGINAL ARTICLE Simple method for determination of oxalic acid in forages using high-performance liquid chromatography Mohammad Mijanur Rahman 1 , Mitsuhiro Niimi 2 and Osamu Kawamura 2 1 The United Graduate School of Agricultural Sciences (University of Miyazaki), Kagoshima University, Kagoshima, Japan 2 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan Keywords Forage; high-performance liquid chromatography; oxalic acid. Correspondence Osamu Kawamura, Division of Grassland Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan. Email: kawamura@cc.miyazaki-u.ac.jp This work was presented at the 63rd Meeting of the Japanese Society of Grassland Science (March 2007). Received: 23 November 2006; accepted: 14 June 2007 doi: 10.1111/j.1744-697X.2007.00093.x Abstract We established a simple method for determination of oxalate in forage grasses using high-performance liquid chromatography (HPLC) with an anion analysis column (Shodex 1C SI-90 4E) and sodium hydrogen carbonate (NaHCO 3 ) as eluent. Oxalate was determined after extraction with water or 1 mol HCl (soluble form or total, respectively). Insoluble oxalate was estimated as the difference between total oxalates and soluble form. Recovery of oxalate after HPLC was above 97%. Oxalates in forages could be successfully determined by this method. The method was simple, fast and inexpensive. Introduction Oxalates are widely distributed in plants either in a soluble form of potassium, sodium and ammonium salts or in an insoluble form of calcium and magnesium salts (Holloway et al. 1989). Grazing animals become calcium deficient through eating forages with high concentrations of oxalate salts (Jones et al. 1970; McKenzie et al. 1988). Thus, the oxalate salts reduce the bioavailability of calcium. Recently, various methods have been developed to determine oxalate using amperometry (Leon et al. 1990), chemilumines- cence (Balion and Thibert 1994; Gaulier et al. 1997a, b, 1998; Perez-Ruiz et al. 1999), fluorometry (Perez-Ruiz et al. 1994), spectrophotometry (Infantes et al. 1991; Petrarulo et al. 1994) and gas chromatography (Gelot et al. 1980). Some of these methods are highly sensitive and specific, but are complicated in procedure, time consuming and/or expensive. The high- performance liquid chromatography (HPLC) method has been shown to be accurate and reliable for determinations of oxalate content in plant materials (Wilson et al. 1982; Holloway et al. 1989; Brega et al. 1992; Huang and Tanudjaja 1992; Utzman 1993; Fengwu et al. 1998). The present study dealt with a simplification of the HPLC method to determine oxalates in forages. Materials and methods Sample preparation Forages were dried at 70°C for 48 h in a forced-air oven, and ground to pass through a 1-mm screen using a Wiley mill. The ground samples were stored in screw-topped plastic containers at room temperature. Extraction of oxalate Soluble and total oxalates in the sample (0.5 g) were extracted with 15 mL of distilled water and 1 mol HCl, respectively (Libert and Franceschi 1987). The suspension of sample was heated in a bath of boiling water for 18 min (Huang and Tanudjaja 1992). After cooling to room temperature, the mixture was filtered through filter paper (5A; Toyo Roshi Kaisha, Tokyo, Japan), washed with water and made up to 50 mL.