HPLC method for the assessment of tryptophan metabolism utilizing separate internal standard for each detector

The development of a validated method, applicable for the measurement of tryptophan (TRP) and serotonin (5-HT), and that of the neuroprotective branch of the kynurenine pathway from several different biological matrices, including mouse brain, is described. Following the spectral analysis of the metabolites, they were quantified with reversed-phase high-performance liquid chromatography (HPLC), using separate internal standards (ISs) for UV (3-nitro-L-tyrosine) and fluorescent (the newly utilized 4-hydroxyquinazoline-2-carboxylic acid) detectors. With regard to validation parameters, selectivity, linearity, limit of detection, limit of quantification, precision and recovery were determined. Although the linearity ranges were different for the assessed matrices, the correlation coefficient was >0.999 in each case. Furthermore, good intra- and inter-day precision values were obtained with coefficient of variation