Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography–mass spectrometry

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography–electrospray ionization mass spectrometry (LC–MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-2H4) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C18 cartridge prior to injection onto a gradient eluted Hypurity C18 reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6–8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n = 10) and <6.0% (n = 9), respectively. The new LC–MS method was highly correlated with an established GC–MS method for urinary 5-HTOL (r2 = 0.99, n = 70; mean 5-HTOL/GTOL ratio = 1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.