Development, validation and clinical evaluation of a dried urine spot method for determination of hippuric acid and creatinine Marina Venzon Antunes ⁎, Camila Ghani Niederauer, Rafael Linden Institute of Health Sciences, Universidade Feevale, Novo Hamburgo, Brazil abstract article info Article history: Received 11 April 2013 Received in revised form 1 July 2013 Accepted 1 July 2013 Available online 15 July 2013 Keywords: Dried urine spots Hippuric acid Creatinine HPLC Objectives: The purpose of this study was to develop and validate a HPLC–DAD method for determination of creatinine and hippuric acid in dried urine spots (DUS), evaluating its clinical applicability. Design and methods: Sample preparation was based on a simple one step extraction with water. Analy- sis was performed in a reversed phase column Hypersil Gold® C18 (150 × 4.6 mm, 3 μm) with isocratic elu- tion. Mobile phase was a mixture of triethylammonium phosphate buffer 5 mM, pH 3.3 and acetonitrile (90:10, v/v). Results: Total analytical run time was 6 min. Precision assays presented CV% lower than 11.8%. Accuracy was 96.0–103%. The lower limit of quantitation was 50 mg L -1 . Mean extraction yields were 99% for CR and 79.3% for HA. Analytes were stable in DUS up to 11 days at 40 °C. The method was tested in 49 paired DUS and urine samples, with acceptable agreement. HA–CR concentration ratios in DUS were in the range of 0.06 to 2.74, being in average 100.7% from those values found in urine. Conclusions: DUS samples are a useful alternative for biological monitoring of toluene occupational ex- posure, especially in Developing Countries where sample logistics could be an important concern. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. 1. Introduction Toluene is an aromatic hydrocarbon solvent widely present in sev- eral occupational settings, as in the production of paints, adhesives, plastic materials, among many others. Occupational exposure to tolu- ene is associated to neurotoxic, hepatotoxic and nephrotoxic effects, and exposed workers must be constantly monitored [1]. Toluene is eliminated in urine mainly in the form of polar metabolites. Biotrans- formation of toluene includes an initial conversion to benzylic alcohol by cytochrome P450 oxidases, followed by further oxidation to benz- aldehyde and benzoic acid. Benzoic acid is conjugated with glycine to form the major toluene metabolite, hippuric acid (HA) [2]. HA concentrations in urine are correlated with environmental concentrations of toluene, being considered an adequate marker for monitoring the occupational exposure to this compound [3]. Howev- er, significant amounts of HA can be derived from the intake of a diet containing benzoic acid or benzoate preservatives. Food associated to an increase in urinary HA includes peaches, green coffee beans, fruit juices, sodas, industrialized bread, ketchup, mustard, and several others [4]. The Brazilian occupational regulations require that workers ex- posed to toluene must be subjected to frequent biological monitoring. To this purpose, HA as well as creatinine (CR) must be measured at an end-of-shift sample, with a maximum permitted HA/CR ratio of 2.5 g (Brasil, Norma Regulamentadora 7, 1994). Therefore, an analytical method that allows the simultaneous determination of HA and CR are particularly useful, representing a significant advantage in terms of time and resources [3]. Currently, there is no immunochemical method for HA commer- cially available, what requires the use of chromatographic methods, usually performed in specialized laboratories. Therefore, sample transportation is usually necessary and sample stability is a major issue, especially when samples must be transported over large dis- tances under warm weather. Water is the medium for many chemical decomposition reactions. As a consequence, urine samples require re- strictive storage and transportation conditions [5]. An alternative to urine samples in biomonitoring is the use of dried urine spots on paper (DUS), which allows the stabilization of the analytes by drying, as well as logistics savings due to the small volume to be transported and to the possibility to keep the specimens at room temperature [6,7]. To date, there is only one report of the use of DUS for the determi- nation of HA and CR, published by Ogata and Taguchi. The authors de- scribed that both compounds were stable when stored at 25 °C for two weeks and when stored at 0 °C for one month [8]. However, no validation data was presented and no comparison between DUS and urine was performed. Considering the potential applicability of DUS for the simulta- neous measurement of HA and CR, especially in Developing Coun- tries, the purpose of this study is to develop and validate a method, Clinical Biochemistry 46 (2013) 1276–1280 ⁎ Corresponding author at: Universidade Feevale, ERS 239 Km nº 2755, 90430-001 Novo Hamburgo, RS, Brazil. Fax: +55 51 35868800. E-mail address: marinaantunes@feevale.br (M.V. Antunes). 0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.07.004 Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem