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

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Abstract
Using native fluorescence detection, 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and tryptophan were resoved 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 pH5 and 6 the compounds were well resolved from each other. The limit of quantitatiave detection of 5-HT and 5-HIAA was 3.5 nmol/L. The overal 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)μmol/L, (n=12). Whole blood 5-HT analysis is unreliable in comparison with platelet-poor plasma.