Development, Validation and Clinical Evaluation of a Dried Urine Spot Method for Determination of Hippuric Acid and Creatinine

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. Analysis was performed in a reversed phase column Hypersil Gold C18 (150 × 4.6 mm, 3 μm) with isocratic elution. 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 exposure, especially in Developing Countries where sample logistics could be an important concern.