Journal of Chromatography B, 805 (2004) 347–351 Short communication High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine Julia B. Ewaschuk a , Jonathan M. Naylor b , Wade A. Barabash a , Gordon A. Zello a,∗ a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9 b Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B4 Received 15 August 2003; received in revised form 2 March 2004; accepted 2 March 2004 Available online 8 April 2004 Abstract To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of d-(-) and l-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and dl-(±)-lactic acids in calf feces and dl-(±)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 m octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-l-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2–14.8mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for d- and l-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for dl-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis. © 2004 Elsevier B.V. All rights reserved. Keywords: Enantiomer separation; Lactic acid; Pyruvic acid; Acetic acid 1. Introduction The majority of HPLC methods available for the analysis of organic acids and the separation of d-(-)- and l-(+)-lactic acid have been validated for aqueous media, and in a few cases, for use in serum [1–4]. There is a need to better un- derstand the etiology of metabolic acidosis associated with diarrhea, as d-(-)-lactic acid has been recently reported to contribute significantly to the drop in blood pH observed in calves with severe diarrhea [5]. Organic and lactic acid mea- surements in less common biological fluids would improve the understanding of metabolic disturbances associated with diarrhea. To analyze lactic acid enantiomers in more com- plex biological fluids such as feces or urine, the only avail- able method is enzymatic, using d-(-)- or l-(+)-lactic acid dehydrogenase. The enzymatic method, however, is subject ∗ Corresponding author. Tel.: +1-306-966-5825; fax: +1-306-966-6377. E-mail address: zello@sask.usask.ca (G.A. Zello). to various sources of error [6]. Therefore, the development and validation of a stereoselective HPLC method is required for the accurate measurement of lactic acid enantiomers in these biological matrices. To facilitate further investigation of metabolic acidosis in diarrhea, the objective of this study was to adapt and validate our previously reported HPLC assays [7] for the analysis of lactic acid enantiomers and other organic acids to feces, rumen fluid and urine. Modifications to the origi- nal method were required, specifically to sample collection, sample preparation methods and selection of a different in- ternal standard for the non-stereoselective assay. 2. Experimental 2.1. Chemicals and equipment l-(+)-Lactic, lithium d-(-)-lactic, acetic, pyruvic, mal- onic and adipic acids were purchased from Sigma (St. Louis, 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.03.004