Optimization of conditions for the simultaneous separation of ten tryptophan metabolites using reversed-phase high-performance liquid chromatography

Rapid Isocratic Liquid Chromatographic Separation and Quantification of Tryptophan and Six kynurenine Metabolites in Biological Samples with Ultraviolet and Fluorimetric Detection

“…Most current analytical procedures involve relatively long runs of between 15 and 70 min for Trp and a small number of metabolites (up to 4), 2–7 or 17–40 min for a larger number (6–9). 8–11 In more recent studies using LC-MS, faster separations were achieved. Thus, using solid-phase extraction, Trp and 2 kynurenines were separated in 8 min, 12 whereas in another study, 13 Trp, 6 kynurenines and 9 other compounds were separated in 5 min.…”

“…These preliminary observations strongly suggest that it is possible to develop this method further to include QA (currently best determined by GC-MS 19 ), other kynurenine pathway metabolites (eg, picolinic, nicotinic and quinaldic acids) and indole metabolites, although a longer run time may be necessary. Such separation has already been achieved using gradient elution over 18-50 min 9,10 and it therefore remains to be seen if this can be achieved isocratically in a shorter run. Our rapid isocratic procedure was subjected to a number of validation criteria.…”

“…For example, current HPLC methods can determine between 1 and 4 Trp metabolites in serum or urine in 15-70 min [2][3][4][5][6][7] and, although faster methods have been developed for analysis of up to 6-9 kynurenine metabolites, a run still takes 17-40 min and most require additional pre-column sample deproteinisation and clean-up, and the use of gradient elution. [8][9][10][11] More recently, HPLC-MS has been used for more rapid separation of kynurenines in two studies. In one, 12 only Trp, kynurenine and 3-hydroxykynurenine were separated within 8 min using on-line solid-phase extraction, whereas in the other, 13 Trp, 6 kynurenines, B-vitamin markers and others (in total 16 analytes) were separated within 5 min.…”

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

“…Although several methods have been developed for the urinary metabolic acids in the tryptophan-NAD pathway [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24], they are either limited to the analysis of a single acid in urine samples or to the instrumental separation and detection only. The objective of this study was to develop a LC/MS based method for the concurrent measurement of QA, PA and NA in urine samples.…”