Determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and tryptophan in plasma and urine by HPLC with fluorimetric detection.
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.
Bearcroft
C P
CP
Digestive Diseases Research Centre, Medical College, St Bartholomew's Hospital, London, UK.
Farthing
M J
MJ
Perrett
D
D
D001792:Blood Platelets / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006897:Hydroxyindoleacetic Acid / Q000032:analysis* / Q000097:blood / Q000652:urine; D010949:Plasma / Q000737:chemistry; D012701:Serotonin / Q000032:analysis* / Q000097:blood / Q000652:urine; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis* / Q000097:blood / Q000652:urine
D012701:Serotonin; D006897:Hydroxyindoleacetic Acid; D014364:Tryptophan