An analytical method for the measurement of acid metabolites of tryptophan-NAD pathway and related acids in urine

An analytical method has been developed for the measurements of five urinary acids namely, quinolinic acid, picolinic acid, nicotinic acid, 2-pyridylacetic acid and 3-pyridylacetic acid. The high performance liquid chromatograph–electrospray ionization mass spectrometry was operated in positive polarity under selected ion monitoring mode, with a column flow rate of 0.2 ml/min and an injection volume of 20 μl. The method used isotope-labelled picolinic acid (PA-d4) and nicotinic acid (NA-d4) as internal standards for the quantification. The sample preparation involved parallel use of two different types of mixed-mode solid phase extraction cartridges (Strata-X-AW for the extraction of quinolinic acid, and Strata-X-C for the remaining acids). Quantitative analysis of five target acids in several human and rat urine samples showed that the levels of acids were relatively uniform among rats while larger variations were observed for human samples.