Determination of 5‐hydroxytryptamine, 5‐hydroxyindoleacetic acid and tryptophan in plasma and urine by HPLC with fluorimetric detection

Abstract

Using native fluorescence detection, 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and tryptophan were resolved from themselves and other naturally occurring compounds using reversed-phase HPLC within 5 min. Deproteinated platelet-poor plasma (PPP) and crude diluted urine were injected directly into the chromatograph. Careful selection of the HPLC column is important and various octadecyl silica (ODS) and base deactivated silic (BDS) columns were evaluated. Pre-treatment of an ODS column with tetrabutylammonium ions gave good selectivity. Between pH 5 and 6 the compounds were well resolved from each other. The limit of quantitative detection of 5-HT and 5-HIAA was 3.5 nmol/L. The overall chromatogram obtained using native fluorescence is cleaner than that obtained with the more commonly employed electrochemical (EC) systems although the chromatography is effectively the same. For analysis of 5-HT in plasma, collection in EDTA was more efficient than lithium heparin. Plasma 5-HT in healthy volunteers was mean 61 (SD = +/- 73) nmol/L, n = 20; urine 5-HIAA gave mean 28.95 (SD = +/- 0.98) mumol/L, (n = 12). Whole blood 5-HT analysis is unreliable in comparison with platelet-poor plasma.

Determination of 5-Hydroxyindole-3-acetic Acid in Human Urine by High-performance Liquid Chromatography with Chemiluminescence Detection

“…This sensitivity is much higher than that of HPLC with native fluorescence [1][2][3] and with electrochemical detection, [4][5][6][7][8] and is comparable to that of HPLC with fluorescence derivatization. [9][10][11][12][13] However, the large peak (peak 3′ in Fig.…”

“…The mean value and the standard deviations (40.1 ± 6.4 nmol/ml) were in good agreement with those obtained by other workers. [1][2][3][4][5][6][7][8] On the other hand, the concentrations of 5HIAA for the carcinoid patients (103.8 and 158.3 nmol/ml) were significantly greater than those obtained for healthy volunteers.…”

“…Various high-performance liquid chromatographic (HPLC) methods developed for the determination of 5HIAA in human urine have incorporated fluorometric [1][2][3] and electrochemical [4][5][6][7][8] determinations based on the native fluorescence and oxidation of the substance, respectively. However, these detection methods are not very sensitive and/or selective for 5HIAA.…”

The inhibition of cholera toxin‐induced 5‐HT release by the 5‐HT3 receptor antagonist, granisetron, in the rat

“…5-HT and tryptophan levels were determined by high performance liquid chromatography and¯uorimetric detection using an ODS column (10064.6 mm, Hichrom Ltd, Theale, Reading, Berkshire) (Bearcroft et al, 1995). The column was eluted with an LKB 2150 pump set at 1 ml min 71 , ambient temperature.…”

Simultaneous Determination of Serotonin and 5-Hydroxyindole-3-acetic Acid in Human Urine by Automated Precolumn Derivatization and Semi-microbore Column Liquid Chromatography with Fluorescence Detection

“…Various high-performance liquid-chromatographic (HPLC) methods, including fluorometric 7,8 and electrochemical detection, [9][10][11] have been described for the determination of 5-HT or 5-HIAA in urine. However, the majority of them lack sensitivity and specificity, or require complicated pretreatments, such as liquid-liquid extraction 8 and solid-phase extraction 9,11 or column-switching 10 for the determination of these compounds in biological samples, since the crude sample contains many fluorescent and electrochemically active compounds that otherwise would interfere with the analysis.…”